Cyclic Adenosine 3',5'-Monophosphate(cAMP)/cAMP-Responsive Element Modulator (CREM)-Dependent Regulation of Cholesterogenic Lanosterol 14
-Demethylase (CYP51) in Spermatids
Damjana Rozman,
Martina Fink,
Gian Maria Fimia,
Paolo Sassone-Corsi and
Michael R. Waterman
Institute of Biochemistry (D.R., M.F.) Medical Center for
Molecular Biology Medical Faculty University of Ljubljana 1000
Ljubljana, Slovenia
Institute de Génétique et de
Biologie Moléculaire et Cellulaire (G.M.F., P.S.-C.) Centre
Nationale de la Recherche Scientifique-INSERM-ULP 67404
Illkrich Strasbourg, France
Department of Biochemistry
(M.R.W.) Vanderbilt University School of Medicine Nashville,
Tennessee 37232-0146
 |
ABSTRACT
|
---|
Lanosterol 14
-demethylase (CYP51) produces MAS
sterols, intermediates in cholesterol biosynthesis that can reinitiate
meiosis in mouse oocytes. As a cholesterogenic gene, CYP51 is regulated
by a sterol/sterol-regulatory element binding protein (SREBP)-dependent
pathway in liver and other somatic tissue. In testis, however,
cAMP/cAMP-responsive element modulator CREM
-dependent regulation
of CYP51 predominates, leading to increased levels of shortened CYP51
mRNA transcripts. CREM-/- mice lack the abundant germ cell-specific
CYP51 mRNAs in testis while expression of somatic CYP51 transcripts is
unaffected. The mRNA levels of squalene synthase (an enzyme preceding
CYP51 in cholesterol biosynthesis in testis of CREM-/- mice are
unchanged as compared with wild-type animals, showing that
regulation by CREM
is not characteristic for all
cholesterogenic genes expressed during spermatogenesis. The
-334/+314 bp CYP51 region can mediate both the sterol/SREBP-dependent
as well as the cAMP/CREM
-dependent transcriptional activation.
SREBP-1a from somatic cell nuclear extracts binds to a conserved
CYP51-SRE1 element in the CYP51 proximal promoter. The
cAMP-dependent transcriptional activator CREM
from germ cell
nuclear extracts binds to a conserved CYP51-CRE2 element while no
SREBP-1 binding is observed in germ cells. The two regulatory pathways
mediating expression of CYP51 describe this gene as a cholesterogenic
gene (SREBP-dependent expression in liver and other somatic cells) and
also as a haploid expressed gene (CREM
-dependent expression in
haploid male germ cells). While in somatic cells all genes involved in
cholesterol biosynthesis are regulated coordinately by the
sterol/SREBP-signaling pathway, male germ cells contain alternate
routes to control expression of cholesterogenic genes.
 |
INTRODUCTION
|
---|
Cholesterol biosynthesis is a housekeeping pathway believed to
take place in virtually all cells in mammals. The presqualene portion
of this pathway is particularly well characterized since all enzymes
have been studied independently, and the coordinate regulation of the
genes encoding these enzymes by a sterol-dependent mechanism is
established (1). Lanosterol 14
-demethylase (CYP51, P45014DM) is a
cytochrome P450 enzyme involved in the postsqualene portion of
cholesterol biosynthesis. It is encoded by the CYP51 gene, the most
evolutionarily conserved gene in the large cytochrome P450 superfamily
(2). The postsqualene portion of cholesterol synthesis consists of at
least eight enzymatic steps and only certain of the corresponding genes
have been cloned (3, 4, 5, 6). CYP51 removes the 14
-methyl group from
lanosterol producing the 14-demethylated sterol FF-MAS (follicular
fluid meiosis-activating sterol). FF-MAS is further metabolized by
sterol 14-reductase to produce T-MAS (testis meiosis activating sterol)
(Fig. 1
). Unlike the presqualene portion
of the pathway where intermediates have well known functions in
additional biological processes (i.e. farnesylation of
proteins, dolichol, heme A synthesis), the postsqualene portion of the
pathway has been considered as exclusively dedicated to cholesterol
biosynthesis. Recently, however, FF-MAS and T-MAS were found to
accumulate in ovary or testis and to have the capacity to reinitiate
meiosis in an in vitro mouse oocyte assay (7, 8). Being
intermediates of cholesterol biosynthesis, MAS sterols are not detected
in most tissues, but are easily observed in gonads (7, 9). These
findings suggest the existence of tissue-specific regulatory
pathways mediating expression of genes involved in MAS sterol
production.

View larger version (17K):
[in this window]
[in a new window]
|
Figure 1. Sterol Flux of the Postsqualene Part of Cholesterol
Biosynthesis
Lanosterol (lanosta-8,24-diene-3ß-ol), FF-MAS
(4,4-dimethyl-5 -cholesta-8, 14,24-triene-3ß-ol) was isolated from
human follicular fluid (7 ) and can be detected also in testis (45 ).
T-MAS (4,4-dimethyl-5 -cholesta-8,24-diene-3ß-ol) was isolated from
bull testis (7 ) and is detected also in ovary (M. Baltsen and
A. G. Byskov, personal communication).
|
|
CYP51 is ubiquitously expressed due to its role in cholesterol
biosynthesis. The highest level of CYP51 mRNA is found in testis where
a shortened testis-specific transcript is observed (3, 10). The rat
testis-specific CYP51 mRNA is shortened in the 3'-untranslated
region being restricted to postmeiotic germ cells, spermatids of
stages VIIXIV (10). This transcript arises from the same promoter as
longer (somatic) rat CYP51 transcripts, using an upstream
polyadenylation site that is not used in somatic cells (11).
Transcriptions of human (12) and rat (11) CYP51 genes begin at multiple
locations, as is characteristic for housekeeping genes. Within the
GC-rich promoter, putative sterol-regulatory element (SRE), and
cAMP-response element (CRE) motifs are found (12). The presence of
SRE-like motifs suggests regulation by oxysterols via a
mechanism similar to that associated with genes involved in the
presqualene portion of cholesterol biosynthesis and in cholesterol
homeostasis (1). The presence of putative CREs suggests that a
cAMP-dependent signaling pathway could also be involved in regulation
of the CYP51 gene.
Herein we report that lanosterol 14
-demethylase follows the pattern
of cholesterogenic gene expression in somatic cells and the pattern of
haploid gene expression in spermatids. Haploid male germ cells seem to
contain alternative routes to mediate expression of certain
cholesterogenic genes. While increased expression of CYP51 in
spermatids is mediated by binding of the cAMP-dependent transcriptional
activator CREM
(cAMP response element modulator-
) to CYP51-CRE2
and no SREBP (sterol response element binding protein) binding is
observed, the expression of squalene synthase, another enzyme of
cholesterol biosynthesis is independent of CREM
in spermatids.
 |
RESULTS
|
---|
CYP51 Is a Cholesterogenic Gene Regulated by SREBPs in Somatic
Cells
The coordinate regulation of the presqualene portion of
cholesterol biosynthesis is well established. CYP51, the first member
of the postsqualene portion of this pathway to be studied is also
regulated by this mechanism. TgSREBP-1-a(460) transgenic mice
expressing the mature form of the transcription factor SREBP-1a in
liver show a 4.6-fold increased expression of mouse CYP51 mRNA
transcripts (Fig. 2
). Two SRE-like
sequences similar to the SREBP recognition site ATCACCCCAC in the
low-density lipoprotein (LDL) receptor promoter are present in the
sense orientation in the 5'-part of the human CYP51 gene (12). One of
the SRE-like motifs (ATCACCTCAG; CYP51-SRE1; Fig. 3
) is 80% identical to the functional
SRE-1 element in the LDL promoter gene (ATCACCCCAC) and is 100%
conserved between human (12) and rat (11). The other sense human
SRE-like element (GGCACCCCGC; CYP51-SRE2; Fig. 3
) is not found in the
rat CYP51 gene (11). The purified transcription factor SREBP-1a binds
only to CYP51-SRE1 but not to CYP51-SRE2 (Fig. 4A
). SREBP-1 from nuclear extracts of
cell lines derived from human liver (HepG2), human adrenal (H295R), and
human choriocarcinoma (JEG-3) cells binds specifically to the conserved
CYP51-SRE1 as determined by supershift and competition studies. For
example, CYP51-SRE1-bound SREBP proteins from H295R cells (Fig. 4B
, lane 3) give a higher mol wt complex (supershift) in the presence of
the crude anti-SREBP-1 (Fig. 4B
, lane 2), and the binding is competed
with excess of unlabeled CYP51-SRE1 probe (Fig. 4B
, lane 5). Similarly,
a SREBP protein complex is formed with CYP51-SRE1 using JEG-3 nuclear
extracts (Fig. 4C
). Anti-CREB (cAMP-response element binding protein)
antibody doesnt shift this complex (Fig. 4C
, lane 1), while purified
anti-SREBP-1 disrupts the complex (Fig. 4C
, lane 5), and unlabeled
CYP51-SRE1 competes the binding (Fig. 4C
, lane 4).

View larger version (29K):
[in this window]
[in a new window]
|
Figure 2. A Representative Picture of CYP51 mRNA Expression
in the Pooled Liver RNAs from Wild Type (w.t.) and TgSREBP-1a (460)
Transgenic Mice Overexpressing the Mature Form of the Transcription
Factor SREBP-1a in Liver (22 )
Northern analysis was performed with partial mouse CYP51 cDNA as a
probe. After correction for unequal loading using
glyceraldehyde-3-phosphate dehydrogenase hybridization, the fold
increase is 4.6 as determined by the phosphoimager. The experiment was
performed two times with identical outcome.
|
|

View larger version (41K):
[in this window]
[in a new window]
|
Figure 3. Human CYP51 5'-Flanking Region
Potential SREs, CREs, and GC box are marked. CYP51-SRE2 does not bind
SREBP-1a and is most probably not a functional SRE element.
Arrows indicate transcription start sites (12 ). +1
indicates the major transcription start site.
|
|

View larger version (33K):
[in this window]
[in a new window]
|
Figure 4. Binding of SREBP-1a to CYP51-SRE Elements
A, Binding of E. coli expressed SREBP-1a to the SRE
element of the LDL receptor gene (lane 1, control) and to two SRE
elements of the human CYP51 gene (lanes 2 and 3). B and C, Binding of
SREBP-1a in nuclear extracts of human cell cultures to CYP51-SRE1. B,
Nuclear extracts from HepG2 cells (lane 1) and H295R cells (lanes
25). Three microliters of the crude anti-SREBP-1 antibody were added
(lane 2). The CYP51-SRE1 competitior (lanes 4 and 5) was added at the
molar ratios indicated. C, Nuclear extracts from JEG-3 cells. Lane 1, 2
µl of the anti-CREB antibody; lane 3, 1000 x molar excess of
the unspecific competitor; lane 4, 1000 x molar excess of
specific competitor; lane 5, 2 µl of the purified anti-SREBP-1
antibody.
|
|
CYP51 Is a Haploid-Expressed Gene Regulated by CREM
in
Spermatids
In situ hybridization of mouse testis showed that CYP51
mRNA is abundantly present in the seminiferous epithelium (Fig. 5B
). Only background levels of CYP51 mRNA
are detected in steroidogenic Leydig cells while strong signals are
observed in these cells with a probe for mRNA encoding the
steroidogenic enyzme 17
-hydroxylase/17,20-lyase cytochrome P450
(CYP17, Fig. 5E
). The intensity of the CYP51 signal varies greatly
between different seminiferous tubule cross-sections (Fig. 5B
),
suggesting stage-specific expression. Spermatogenesis in mouse
seminiferous tubules can be divided into 12 stages of cellular
associations, which are identified by their morphology and the
presence/location of distinct germ cells in each stage (13). Using the
mouse CYP51 antisense riboprobe, only background signal is detected in
stages I-III of the mouse seminiferous wave (Fig. 5D
). In contrast, the
signal is very strong in stages VIIVIII and is restricted to late
round and early elongating spermatids immediately underneath the most
mature elongated spermatids, which face the lumen (Fig. 5
, C and D). It
is clear that the CYP51 signal is largely absent from the cell layer
containing the most mature elongating spermatids that are ready to be
released to the lumen and whose condensed nuclei form a narrow band
lining the lumen (Fig. 5
, C and D). The pattern of CYP51 expression in
mouse testis seems to be identical to that observed in rat (10).

View larger version (168K):
[in this window]
[in a new window]
|
Figure 5. In Situ Hybridization of Adult Mouse
Testis
A and C, Periodic acid Schiff staining; Probes: CYP51 antisense
(B and D); CYP17 antisense (E); CYP17 sense (F). Bar, 400 µm (A, B,
D, and F); bar, 50 µm (C and D); stages of spermatogenesis are marked
with Roman numerals.
|
|
Overexpression of the CYP51 mRNA in late round and early elongating
spermatids of both rodent species matches with the timing of the
highest level of expression of the haploid germ cell-specific
transcription activator CREM
(L. Monaco, P. Sassone-Corsi,
unpublished data). This suggested a role for CREM
in CYP51
transcription during spermatogenesis. To prove this, expression of
CYP51 has been monitored in mice with the disrupted CREM gene (15). The
expression of CYP51 mRNA in liver (Fig. 6A
) is not affected by the absence of
CREM and is controlled by SREBP-1a as shown in Fig. 2
. The expression
of the two abundant CYP51 transcripts in testis depends on the presence
of at least one functional CREM gene. These transcripts in wild-type
and CREM+/- mouse testis are analogous to the shortest CYP51
transcript in rat (10) expressed only in postmeiotic germ cells. The
testis of CREM-/- mice lack the shortest CYP51 mRNAs and contain only
the somatic CYP51 transcripts expressed to similar levels as in liver
(Fig. 6A
). This shows that haploid expression of CYP51 in mouse testis
is CREM
-dependent in vivo.

View larger version (72K):
[in this window]
[in a new window]
|
Figure 6. A Representative Northern Blot Showing Expression
of CYP51 (A) and Squalene Synthase (B) in Liver and Testis of Wild Type
(+/+), CREM Heterozygous (+/-) and CREM Knockout (-/-) Mice
|
|
To evaluate whether CREM
is involved in mediating expression of
other cholesterogenic genes, expression of squalene synthase was
monitored in CREM-/- mice. We have shown previously that in addition
to CYP51, transcription of squalene synthase is also up-regulated in a
stage-specific manner during spermatogenesis (10). Increased levels of
squalene synthase mRNA were observed before meiosis in pachytene
spermatocytes (10) where the activator transcription factor CREM
is
not yet present (16). Monitoring expression of squalene synthase in
CREM-/- mice showed that expression of this early cholesterogenic
gene is not controlled by the cAMP/CREM
mechanism. Similar levels of
squalene synthase mRNA are present in testis of normal, CREM-/+, and
CREM-/- mice (Fig. 6B
). Also, no CRE is found in the 5'-flank of the
human squalene synthase gene (17).
CREM
Binds to the CYP51-CRE Elements Residing in the Promoter of
CYP51
The human CYP51 gene contains two potential cAMP-response elements
(CYP51-CRE1 and CYP51-CRE2) upstream of the major transcription start
site (Fig. 3
). We wanted to evaluate whether purified CREM
or
CREM
present in nuclear extracts of testis and germ cells can bind
to these CYP51-CREs. Proteins in nuclear extracts from whole rat testis
(somatic cells and germ cells) bind strongly to human CYP51-CRE1 (Fig. 7A
, lane 1). Both anti-CREB (Fig. 7A
, lane 2) and anti-CREM (Fig. 7A
, lane 3) antibodies disrupt this gel
shift pattern, giving different supershifts. Thus, it appears that
isoforms of both CREM and CREB proteins can bind to CYP51-CRE1. Binding
is competed with the consensus CRE from the somatostatin gene, showing
that most of the strong signal belongs to CRE-binding proteins (Fig. 7B
).

View larger version (55K):
[in this window]
[in a new window]
|
Figure 7. Binding of Rat Testis cAMP-Response Element Binding
Proteins to the Human CYP51-CRE1 Element
A, Supershift analysis. Antibodies were added to the adult rat testis
nuclear extract: 1, no antibody; 2, anti-CREB antibody (1 µl); 3,
anti-CREM antibody (2 µl). The gel was run at +4 C. B, Competition
analysis: different amounts of the cold CRE consensus probe
(Promega Corp.) were added to the
[32P]-labeled CYP51-CRE1 probe.
|
|
Proteins from rat testis nuclear extracts also bind to human CYP51-CRE2
(Fig. 8
), albeit more weakly than to
CYP51-CRE1. The data show that CREM isoforms from the rat testis and
germ cell nuclear extracts efficiently bind to the CYP51-CRE2 sequence.
The anti-CREM antibody supershifts the complex in testis (Fig. 8A
, lane
5) and germ cells (Fig. 8B
, lane 5), while the anti-CREB antibody
doesnt influence nuclear protein binding to CYP51-CRE2 (Fig. 8
, A and
B, lanes 2 and 3). The anti-CREM antibody has a 100-fold higher
affinity for CREM than for CREB (16). The E. coli expressed
CREM
-protein binds to CYP51-CRE1, CYP51-CRE2, and
CYP51-CRE2new elements, yet with a lower affinity than to
the consensus CRE TGACGTCA (Fig. 8C
). CYP51-CRE2 and
CYP51-CRE2new differ in two bases flanking the CRE octamere
TGACGCGA.

View larger version (39K):
[in this window]
[in a new window]
|
Figure 8. CREM Isoforms of the Adult Rat Testis (A) and Germ
cells (B) Bind to CYP51-CRE2
Supershift or competition analyses were performed by adding antibodies
or competitors to the whole testis (A) or germ cell (B) nuclear
extracts. 1, No antibody; 2, anti-CREB antibody (3 µl); 3, anti-CREB
antibody (5 µl); 4A, 500 x molar excess of CYP51-CRE2; 5,
anti-CREM antibody (3 µl). C, Binding of the E. coli
expressed CREM to CYP51-CRE1 and CYP51-CRE2 elements and to
consensus CRE from the somatostatin gene. CYP51-CRE2new was
checked for binding of CREM after discovery of a sequencing error in
the region flanking the conserved CRE2 octamere. CYP51-CRE2 and
CYP51-CRE2new differ in two bases flanking the CRE element
"TGACGCGA".
|
|
CYP51-CRE1 binding proteins are present in nuclear extracts of mature
rat male germ cells (Fig. 9
, lane 3), but
no CYP51-SRE1 binding proteins can be detected in such extracts (Fig. 9
, lane 4). Using nuclear extracts from mature rat testis,
CYP51-SRE1-binding proteins are readily detected because these extracts
are derived from a mixture of Sertoli cells, Leydig cells, and germ
cells (Fig. 9
, lane 2). This pattern is identical to that obtained with
nuclear extracts from human somatic cells (Fig. 4
). Thus, based on gel
mobility shift binding assays, SREBP is not present in the majority of
male germ cell types isolated by the trypsin-DNAse method.

View larger version (58K):
[in this window]
[in a new window]
|
Figure 9. Binding of Rat Testis and Germ Cell Nuclear
Proteins to the Human CYP51-SRE1 and CYP51-CRE1 Elements
Nuclear extracts isolated from: 1 and 2, adult rat testis; 3 and 4,
germ cells from adult rat testis; 1,3-CYP51-CRE1 probe; 2,4-CYP51-SRE1
probe.
|
|
Sterol-Dependent and cAMP-Dependent Regulation of CYP51 Is Mediated
by the Same Promoter
CYP51 has a housekeeping type 5'-flanking region with no TATA and
CAAT boxes and no initiator (Inr) element. Transcription of CYP51
starts at several locations that differ between human (12) and rat
(11). We linked 648 bp of the 5'-flanking GC-rich region of the human
CYP51 gene including 334 bp upstream from the major transcription start
site to the chloramphenicol acetyltransferase (CAT) reporter. This
construct containing CYP51-SRE1 and CYP51-CRE2 elements was checked for
its ability to induce activity of the CAT reporter in JEG-3 cells under
different physiological conditions: sterol deprivation or cAMP
stimulation (Fig. 10
). JEG-3 are human
choriocarcinoma cells that contain the sterol-dependent signal
transduction pathway and also a cAMP-dependent signal transduction
pathway (18). The functional importance of SREBP-1a and CREM
in
CYP51 transcription has been measured by cotransfection experiments in
these cells. The human CYP51 -334/+314 promoter activity is
essentially unchanged (1.4-fold) in delipidated serum (Fig. 10
, lane
2), stimulated 17-fold when activated by the mature form of the
transcription factor SREBP-1a (lane 3), and 18.5-fold when delipidated
serum and SREBP-1a activation are combined. These data show a marked
stimulation of CYP51 promoter activity by transcription factor
SREBP-1a. The same promoter shows stimulation of activity also by the
cAMP-dependent pathway. Activity is stimulated 4.3-fold by forskolin
(lane 6), indicating activation of CYP51 transcription by endogenous
cAMP-responsive element-binding proteins in these somatic cells. The
cAMP-dependent transcription activator CREM
is highly expressed only
in spermatids, the haploid male germ cells (19). Since no spermatid
cell line is available, JEG-3 cells were cotransfected with CREM
to
mimic the physiology of haploid male germ cells. The human CYP51
promoter activity is stimulated 2.6-fold by CREM
(lane 5) and
6.1-fold by the combined action of forskolin and CREM
(lane 7),
showing that CREM
which is the most abundant cAMP-dependent
transcriptional activator in spermatids (19), and a master switch of
postmeiotic gene expression (15, 20) can mediate expression of
CYP51.
 |
DISCUSSION
|
---|
CYP51 encodes lanosterol 14
-demethylase, a ubiquitously
expressed gene of the postsqualene part of cholesterol biosynthesis.
Transcription of this gene in somatic cells (liver, adrenal, placenta)
follows the pattern of coordinate, sterol/SREBP-dependent regulation
characteristic of genes involved in cholesterol biosynthesis and
homeostasis (1, 21). Expression of somatic mouse CYP51 mRNAs is
4.6-fold higher in livers of SREBP-1a(460) transgenic mice, an increase
similar to that of mRNAs for two cholesterol biosynthetic genes before
squalene (hydroxymethylglutaryl-coenzyme A synthase 6.1-fold and
squalene synthase 5.4-fold) but is lower than
hydroxymethylglutaryl-coenzyme A reductase (37-fold), which is the
major regulatory point of cholesterol biosynthesis (22). The CYP51-SRE1
sequence found in the promoter of human and rat CYP51 genes and
recently also in the mouse CYP51 gene (Debeljak and D. Rozman,
unpublished), is not identical to any of the previously established
SREs (23). The sequence ATCACCTCAG (CYP51-SRE1) is 100% conserved in
human, rat, and mouse CYP51 promoters and is readily recognized by the
purified SREBP-1. SREBP-1 does not bind to the second CYP51-SRE-like
element GGCACCCCGC (CYP51-SRE2; Fig. 4A
). We thus believe that
CYP51-SRE1 is a major point of SREBP interaction within the CYP51
promoter and is responsible for the sterol-dependent activation of
CYP51 transcription as observed in the CAT reporter assay. These data,
together with those in Fig. 10
, strongly indicate that ATCACCTCAG is a
functional SRE sequence.
Surprisingly, the ubiquitously expressed mammalian CYP51 mRNA encoding
cholesterogenic lanosterol 14
-demethylase is expressed to highest
levels in the testis (3, 10). The pattern of CYP51 mRNA expression in
rat (10) and mouse testis (this paper) is very similar, peaking in
round and early elongating spermatids that match the timing of
expression of transcription activator CREM
(L. Monaco and P.
Sassone-Corsi, unpublished data). Germ cells of the testis contain
various activator as well as inhibitory CREB and CREM transcription
factors that arise by differential splicing of both genes (24, 25, 26).
CREM expression is developmentally switched during spermatogenesis from
antagonist forms (CREM
, CREMß, CREM
) in premeiotic germ cells
to the activator forms (CREM
, CREM
1,
CREM
2) in postmeiotic germ cells (19). CREM
plays a
key role in transcriptional activation in haploid male germ cells and
acts as a master switch responsible for the cAMP-dependent
transcriptional activation of several genes expressed at this specific
time during spermatogenesis (27, 28). The homozygous CREM-/- males
are infertile despite normal mating behavior (15, 29) while the females
remain fertile. The list of CREM
-regulated genes includes genes with
established roles in spermatogenesis, such as protamines 1 and 2,
transition protein 1, calspermine, ACE, etc., and a longer list of
genes having roles in germ cell maturation not yet fully established
(16, 30). Our results show that cholesterogenic lanosterol
14
-demethylase belongs to this group of haploid-expressed
transcripts induced by the transcription activator CREM
. High
expression of CYP51 in spermatids results from the appearance of
shorter, germ cell-specific CYP51 transcripts (Fig. 9
) (10). The two
highly expressed mouse CYP51 mRNAs are absent in the testis of
CREM-/- mice, showing that a cAMP-dependent transcription
activator of the CREM family is needed to promote their synthesis.
In contrast to CYP51, expression of another cholesterogenic gene,
squalene synthase, does not depend on the presence of CREM
during
spermatogenesis. Increased levels of squalene synthase mRNA are
observed before meiosis in pachytene spermatocytes (10) where the
activator transcription factor CREM
is not yet present (16).
Squalene synthase expression in the testis of CREM-/- mice is
identical to that in wild-type animals, showing that CREM
-dependent
regulation is not characteristic for all cholesterogenic genes that are
highly expressed during spermatogenesis. In the absence of evidence for
the presence of sterol responsive element-binding proteins in germ
cells, the mechanism for squalene synthase expression in
spermatogenesis remains an enigma.
We wanted to evaluate which elements of the CYP51 promoter may be
involved in mediating the cAMP/CREM
-dependent expression of CYP51 in
spermatids. CYP51-CRE2 (Fig. 3
) is conserved across species, showing
high homology (7/8) to the aligned sequences of the rat (11) and mouse
(N. Debeljak and D. Rozman, unpublished). CYP51-CRE1 shows a
lower (5/8) evolutionary conservation in these species. Since the
evolutionary conserved CYP51-CRE2 binds CREM proteins present in rat
testis specifically or with a preference over CREB proteins, it is the
major candidate for binding of CREM
in germ cells of different
species in vivo. In accordance to this, transfection assays
(Fig. 10
) show that CYP51 promoter containing only CYP51-CRE2 can
efficiently mediate the CREM
-dependent transcriptional
activation.
It is now established that cAMP regulates expression of a
cholesterogenic gene CYP51 in addition to the sterol type of gene
regulation. Both regulatory processes are mediated by the same
-334/+314 CYP51 promoter as determined by the CAT reporter analysis.
Thus, the availability of transcription factors in various cell types
may differentially control transcription of the CYP51 gene. While
expression of the germ cell-specific CYP51 mRNAs is under CREM
control, the expression of longer CYP51 somatic-type transcripts in
germ cells may be regulated in a housekeeping manner due to the
presence of a GC box that binds ubiquitous transcription factors of the
Sp family. It has recently been established that transcription factor
Sp1 plays a major role in determining expression of lactate
dehydrogenase gene during spermatogenesis (31).
In conclusion, CYP51 produces FF-MAS, an oocyte meiosis-activating
sterol (7), which is an intermediate in the coordinately regulated
housekeeping process of cholesterol biosynthesis. Interestingly, in
haploid male germ cells the cAMP/CREM
-dependent regulation of CYP51
predominates over the sterol/SREBP regulation, which coordinates
cholesterol biosynthesis in somatic cells. The haploid expressed mRNAs
of the CYP51 gene belong to the group of CREM
-regulated transcripts
whose detailed role in spermatogenesis (cholesterol production
vs. FF-MAS production) has yet to be determined. Since
another cholesterogenic gene (squalene synthase) is not regulated by
CREM
and apparently no coordinate-type regulation of cholesterogenic
genes occurs in germ cells, it is likely that the
cAMP/CREM
-dependent induction of CYP51 in spermatids does not serve
increased cholesterol synthesis and may thus be associated with
overproduction of MAS sterols, signaling molecules produced by the
gonads.
 |
MATERIALS AND METHODS
|
---|
Preparation of a Partial Mouse CYP51 cDNA Probe
Primers were designed according to the rat CYP51 cDNA sequence
(32) with the addition of ends complementary to the pDIRECT cloning
vector; sense primer: 5'-AAAAGTTGGGGAGAAAGCGG-3' (331351), antisense
primer: 5'-GATGTCCTGGAGGAATGGT-3' (958978). RT-PCR experiments were
performed by standard methods (33). The 647-bp RT-PCR fragment was
cloned into pDIRECT leading to the recombinant plasmid pM14DM. The
partial mouse CYP51 RT-PCR fragment was sequenced (Epicentre
Technologies Corp., Madison, WI) and showed 93.6% homology to the rat
CYP51 cDNA. This partial CYP51 cDNA insert was cleaved from pM14DM with
restriction enzymes EcoRI and BamHI and used as a
probe in in situ hybridization and Northern analyses.
In Situ Hybridization
Adult mouse testes were rinsed in PBS, pH 7.4, and immersion
fixed overnight in 4% paraformaldehyde in PBS, pH 7.4. The tissue was
dehydrated in ethanol and xylene solutions, embedded in paraffin, cut
into 6-µm thick sections and hybridized with the mouse CYP51 cDNA
probe. Sense and antisense mouse CYP51 riboprobes were prepared from
pM14DM by in vitro transcription with T3 and T7 RNA
polymerases in the presence of [35S]UTP. CYP17 riboprobe
was prepared by in vitro transcription from the cloned mouse
743 bp CYP17 fragment (PCR-Direct cloning system,
CLONTECH Laboratories, Inc., Palo Alto, CA)
(34) and used as a positive control.
Northern Analysis
Five SREBP-1a transgenic mice (22) and five littermate wild-type
controls were placed on a high protein/low carbohydrate diet (No.
5789C-3) from Purina Mills Inc. (St. Louis, MO) containing 71% (wt/wt)
casein and 4.25% (wt/wt) sucrose for 2 weeks before study. All mice
were killed after a 12-h fast in the early phase of the light cycle.
Isolation of total RNA from liver and Northern blot analysis was
performed exactly as described (22) using the mouse CYP51 647-bp RT-PCR
fragment as probe. Liver RNA was pooled from five transgenic animals
and from five wild-type animals. Equal amounts (3 µg) of RNA isolated
from individual animals were mixed in the five-animal pool (total 15
µg of RNA). CYP51 analysis was performed in the laboratory of Drs.
J. L. Goldstein and M. S. Brown on RNAs from the same group
of transgenic animals previously used to study expression of other
cholesterogenic genes (22). The bands detected by Northern analysis
were quantified by exposing the filter to a BAS1000 Phosphor-Imager
Fuji Photo Film Co., Ltd., Tokyo, Japan), and results were
normalized to the signal generated by glyceraldehyde-3-phosphate
dehydrogenase mRNA. The analysis was performed twice with identical
outcome.
Total RNA from the liver and testis of CREM-/-, CREM+/- and the
wild-type mice was prepared (15) and 20 µg of each were used for
Northern analysis. Equal loading was verified by staining the gel with
ethidium bromide. Partial mouse CYP51 cDNA and a partial rat squalene
synthase cDNA were used as probes (10). Northern analysis was performed
in the laboratory of Dr. Paolo Sassone-Corsi on RNA samples isolated
from the same group of mice previously used to study expression of
other genes, with equivalent results obtained from five different sets
of mutant and normal mice (15). CYP51 analysis was performed three
times with identical outcome.
Preparation of Nuclear Extracts from Rat Testis and Germ
Cells
Nuclear extracts were prepared at the same time as cytosolic
protein extracts from sexually mature rats (10). Testes of one adult or
five prepubertal Harlan Sprague Dawley (Indianapolis, IN)
rats were decapsulated and homogenized on ice by 50100 strokes with a
glass-Teflon hand homogenizer in assay buffer (100 mM
K3PO4, 0.1 M dithiothreitol (DTT),
0.1 mM EDTA, 20% glycerol). Homogenates were filtered
through a 50-µm filter and spun for 15 min at 1500 x
g and +4 C. The pellet was resuspended by hand
homogenization in 10 ml of ice-cold buffer A (0.25 M
sucrose, 10 mM potassium phosphate buffer, pH 6.8, 5
mM MgCl2) containing the proteinase inhibitor
mixture of 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml
leupeptin, 0.5 mM phenylmethylsulfonylfluoride, and 0.5
mM DTT. After centrifugation at 1500 x g
for 10 min at +4 C, the resulting pellet was suspended in 1 ml of
ice-cold buffer B (0.25 M sucrose, 20 mM HEPES,
pH 7.9, 0.3 M NaCl, 5 mM MgCl2)
containing the same proteinase inhibitor mixture. It was transferred to
Eppendorf tubes, kept 20 min on ice with occasional
vortexing and spun 10 min in a microcentrifuge at +4 C. The supernatant
was recentrifuged at 100,000 x g at +4 C for 30 min,
aliquoted, and frozen in liquid nitrogen.
Germ cells from groups of two sexually mature male rats were prepared
by mechanical treatment and trypsinization (10). Briefly, decapsulated
testes were thoroughly minced with an array of sealed razor blades and
treated with DNAse and trypsin. This procedure destroys most of the
Sertoli and Leydig cells while germ cells remain intact (35). Germ
cells were pelleted, washed and suspended in 5 ml of assay buffer, and
hand homogenized on ice by 100 strokes. After centrifugation at
1500 x g for 15 min at +4 C, the pellet was suspended
in 10 volumes of ice-cold buffer A and nuclear extracts were
prepared.
Preparation of Nuclear Extracts from Cell Lines
Human adrenocortical cells, NCI-H295R (36, 37), were grown in a
mixture of DMEM/Hams F12 medium/Nuserum IV (45:45:10). Cells were
split 1:2 twice a week. Human hepatoma HepG2 cells and human
choriocarcinoma JEG-3 cells were grown in DMEM with 10% bovine serum.
Cells were split twice a week: HepG2, 1:7 and JEG-3, 1:5. All cultures
were grown at 37 C and 5% CO2. Nuclear extracts were
isolated from two 150-mm culture dishes containing two thirds confluent
cells as described (38). Protein concentrations were determined by the
Bio-Rad dye binding assay according to the manufacturers instructions
(Bio-Rad Laboratories, Inc., Richmond, CA).
Expression and Purification of Recombinant CREM
and SREBP-1a
Proteins
Purification of bacterially expressed CREM
protein was
previously described (16). Recombinant SREBP-1a protein (amino acids
1490) was expressed in E. coli as a 6xHis-tagged fusion
protein at the amino terminus (39) and purified by Ni2+
affinity chromatography by the QIAexpressionist system
(Qiagen, Chatsworth, CA) as recommended by the
manufacturer. The expression plasmid was a gift of T. F. Osborne
(University of California, Irvine, CA). Protein concentrations for both
recombinant proteins were determined by the Bio-Rad Laboratories, Inc. dye binding assay according to the manufacturers
instructions. The homogenity of samples was checked by SDS/PAGE
followed by Comassie blue staining.
Production and Purification of the anti-SREBP-1
The SREBP-1 monoclonal antibody was produced from the IGG-2A4
(CRL 2121) hybridoma cell line (ATCC, Manassas, VA) and
purified by protein A-Sepharose affinity chromatography (Sigma Chemical Co., St. Louis, MO) as described (40). Antibody is
directed against amino acids 301407 of SREBP-1 and therefore
recognizes both SREBP-1a and SREBP-1c isoforms, which differ only in
the sequence of the first exon (41). Isoform 1c is a weaker
transcription activator than isoform 1a in most cell types (42).
Gel Shift Analysis
Oligonucleotides used to generate double-stranded fragments
containing CRE and SRE elements were: CYP51-SRE1
(5'-GGCCGAGATCACCTCAGGCGCT-3' and 5'-GCGAGCGCC TGAGGTGATCTCG-3');
CYP51-SRE2 (5'-CGTGTCCCGGCACCCCGCACCCGG-3' and
5'-TGCCCGGGTGCGGGGTGCCGGGAC-3'); LDL-SRE
(5'-TTTGAAAATCACCC CACTGCA-3' and
5'-GTTTGCAGTGGGGTGATTTTC-3'); CYP51-CRE1
(5'-GGGACGGGGCTGACCTCACC GTCCT-3' and 5'-AGGACGGTGAGGTCAGCCCCGT-3');
CYP51-CRE2 (5'-GCCCCGTTGA CGCGATGTAGGCCGA-3' and
5'-GATCTCGGCCTACATCGCGTCAACGG-3'); CYP51-CRE2new
(5'-GCCCCGCTGACGCGATGTAGGCCGA-3' and 5'-GATCTCGGCCTACATCGCGTCAGCGG).
Oligonucleotides were purified on 20% acrylamide gels. Pairs were
annealed in equimolar concentrations and end labeled with
32P-ATP by T4 polynucleotide kinase. In some experiments
the commercial CREB consensus oligonuleotide (Promega Corp., Madison, WI) was used. Gel retardation assay was
performed as follows: 10 µg of nuclear proteins or 47 µg of the
overexpressed transcription factor were incubated in a 20 µl reaction
volume containing 20 mM HEPES, pH 7.6, 32 mM
KCl, 80 nM EDTA, 8% glycerol, 0.8 mM DTT, 1
µg poly dI-dC, 1 µg yeast tRNA at room temperature for 5 min.
Labeled probe (3 x 104 cpm) was added and incubated
on ice for an additional 10 min. DNA-protein complexes were resolved on
a 5% native acrylamide gel in 0.5 x TBE buffer at room
temperature and detected by autoradiography. For gel supershift
analyses, different volumes of rabbit sera containing the anti-CREM
antibody (16) or anti-CREB antibody were added. Anti-rat CREB antibody
was prepared by injecting rabbits with the E. coli expressed
rat CREB protein by standard procedures (43). Anti-SREBP1 antibody was
used before (crude antibody) or after purification on the protein A
Sepharose.
Preparation of CAT-Reporter Constructs
The 5'-untranslated region (-334/+314) of the human CYP51 gene
was amplified by the cloned Pfu polymerase
(Stratagene) using sense (5'-GGAGGAGGGTGAGGTGCC
ACAGTTCGAGGT-3') and antisense (5'-ACCTCGAACTGTGGCACCTCACCCTTCTCC-3')
primers from the cosmid 121G12 containing the whole human CYP51 gene
(12). The fragment was cloned into the SmaI-digested pCAT
basic plasmid (Promega Corp.). Two independent clones were
completely sequenced.
Cell Culture, Transfections, and CAT Assay
JEG-3 (human choriocarcinoma cells) were cultured in DMEM
supplemented with 10% bovine calf serum and 1%
L-glutamine. Cells were maintained in a humidified
incubator with 5% CO2 at 37 C. They were split 1:3 twice a
week and plated into culture dishes (90 mm diameter) 1 day before
transfection. Cells were transfected at approximately 50% confluency
by the calcium precipitate method (33) with 20 µg of plasmid DNA: 10
µg of the CYP51-CAT construct, 5 µg of the RSVß-gal plasmid (for
normalization), and 3 µg of pSV CREM
or pCMVh SREBP 1a plasmids.
The pCAT basic plasmid was used as the carrier DNA to 20 µg. The CAT
activity of this plasmid is negligible. Twenty four hours after
transfection cells were treated either with forskolin, the artificial
inducer of the cAMP response (25 µM final concentration),
or the medium was removed and changed to DMEM, 10% delipidated serum,
and 1% L-glutamine, which is the media for induction of
the SREBP-dependent response. Cells were harvested 48 h after
transfection (24 h after addition of forskolin or delipidated serum).
CAT assays were performed as described (44). Protein concentrations
were determined by the Bio-Rad dye binding assay according to the
manufacturers instructions (Bio-Rad Laboratories, Inc.).
The ß-galactosidase activity assay was performed as described (33).
CAT activity was normalized by the formula: [CAT activity
(cpm)/ß-gal activity (A420)]/[protein concentration]
(mg/ml). Every transfection was performed at least three times with two
parallel samples in each experiment. The average value and
SEM were calculated with the Excel program (Microsoft Corp.).
Experimental Animals
Animal studies were conducted in accord with the principles and
procedures outlined in Guidelines for Care and Use of Experimental
Animals.
 |
ACKNOWLEDGMENTS
|
---|
We sincerely thank Drs. Jay D. Horton, Michael S. Brown, and
Joseph L. Goldstein from the Department of Molecular Genetics and
Biochemistry, University of Texas Southwestern Medical Center, Dallas,
Texas, for providing data regarding SREBP-1a(460) transgenic mice and
for their critical comments and evaluation of the manuscript. We would
also like to thank Dr. Maria Strömstedt for her help with
preparation of the mouse CYP51 and squalene synthase cDNA probes and
in situ hybridization studies. Thanks also to Patricia Ladd
and Dr. Jesus de Leon (Vanderbilt University, Nashville, Tennessee) for
their assistance in preparing the anti-SREBP-1 antibody and to Dr.
Timothy F.Osborn (Department of Molecular Biology and Biochemistry,
University of California, Irvine, California) for providing the
SREBP-1a protein expression plasmid.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Assistant Professor Damjana Rozman, Ph.D., Institute of Biochemistry, Medical Center for Molecular Biology, Medical Faculty University of Ljubljana, Vrazov trg 2, S1-1000 Ljubljana, Slovenia.
This work was supported by USPHS Grants DK-28350, ES 00267, by Grant
9650310N from the American Heart Association and by Grants Z17225,
J11380, and SLO-USA 0002 from the Ministry of Science of
Slovenia.
Received for publication July 1, 1999.
Revision received July 23, 1999.
Accepted for publication August 6, 1999.
 |
REFERENCES
|
---|
-
Goldstein JL, Brown MS 1990 Regulation of the mevalonate
pathway. Nature 343:425430[CrossRef][Medline]
-
Yoshida Y, Noshiro M, Aoyama Y, Kawamoto T, Horiuchi, Gotoh O 1997 Structural and evolutionary studies on sterol 14-demethylase P450
(CYP51). II. Evolutionary analysis of protein and gene structures.
J Biochem 122:11221128[Abstract]
-
Strömstedt M, Rozman D, Waterman MR 1996 The
ubiquitously expressed human CYP51 encodes lanosterol
14
-demethylase, a cytochrome P450 whose expression is regulated by
oxysterols. Arch Biochem Biophys 329:7381[CrossRef][Medline]
-
Matsushima M, Inazawa J, Takahashi E, Suzumori K, Nakamura Y 1996 Molecular cloning and mapping of a human cDNA (SC5DL) encoding a
protein homologous to fungal sterol-C5-desaturase. Cytogenet Cell Genet 74:252254[Medline]
-
Jbilo O, Vidal H, Paul R, De Nys N, Bensaid M, Silve S,
Carazon P, Davi D, Galiegue S, Bourrie B, Guillemont J-C, Ferrara P,
Loison G, Maffrand J-P, Le Fur G, Casellas P 1997 Purification and
characterization of the human SR 31747A-binding protein. J Biol
Chem 272:2710727115[Abstract/Free Full Text]
-
Moebius FF, Fitzky BU, Lee JN, Paik Y-K, Glossmann H 1998 Molecular cloning and expression of the human delta7-sterol reductase.
Proc Natl Acad Sci USA 95:18991902[Abstract/Free Full Text]
-
Byskov AG, Andersen CY, Nordholm L, Thogersen H, Guoliang X,
Wassman O, Guddal JVAE, Roed T 1995 Chemical structure of sterols that
activate oocyte meiosis. Nature 374:559562[CrossRef][Medline]
-
Grondahl C, Ottesen JL, Lessl M, Faarup P, Murray A, Gronvald
FC, Hegelehartung C, Ahnfeltronne I 1998 Meiosis-activating sterol
promotes resumption of meiosis in mouse oocytes cultured in
vitro in contrast to related oxysterols. Biol Reprod 58:12971302[Abstract]
-
Byskov AG, Baltsen M, Andersen CY 1998 Meiosis-activating
sterols: background, discovery and possible use. J Mol Med 76:818823[CrossRef][Medline]
-
Strömstedt M, Waterman MR, Haugen TB, Taskén K,
Parvinen M, Rozman D 1998 Elevated expression of lanosterol
14
-demethylase (CYP51) and the synthesis of oocyte
meiosis-activating sterols in postmeiotic germ cells of male rats.
Endocrinology 139:23142321[Abstract/Free Full Text]
-
Noshiro M, Aoyama Y, Kawamoto T, Gotoh O, Horiuchi T, Yoshida
Y 1997 Structural and evolutionary studies on sterol 14
-demethylase
P450 (CYP51), the most conserved monooxygenase. I. structural analyses
of the gene and multiple sizes of mRNA. J Biochem 122:11141121[Abstract]
-
Rozman D, Strömstedt, M, Tsui L-C, Scherer SW, Waterman
MR 1996 Structure and mapping of the human lanosterol 14
-demethylase
gene (CYP51) encoding the cytochrome P450 involved in cholesterol
biosynthesis; comparison of exon/intron organization with other
mammalian and fungal CYP genes. Genomics 38:371381[CrossRef][Medline]
-
Russell LD, Ettlin RA, Sinha Hikim AP, Clegg ED 1990 Histological and Histopathological Evaluation of the Testis. Choche
River Press, Clearwater, FL
-
Deleted in proof
-
Nantel F, Monaco L, Foulkes NS, Masquilier D, LeMeur M,
Henriksén K, Dierich A, Parvinen M, Sassone-Corsi P 1996 Spermiogenesis deficiency and germ-cell apoptosis in CREM-mutant mice.
Nature 380:159162[CrossRef][Medline]
-
Delmas V, van der Horn F, Mellström B, Jégou B,
Sassone-Corsi P 1993 Induction of CREM activator proteins in
spermatids: downstream targets and implications for haploid germ cell
differentiation. Mol Endocrinol 7:15021514[Abstract]
-
Guan G, Jiang G, Koch RL, Shechter I 1995 Molecular cloning
and functional analysis of the promoter of the human squalene synthase
gene. J Biol Chem 270:2195821965[Abstract/Free Full Text]
-
Zhou Y, Sun Z, Means AR, Sassone-Corsi P, Bernstein KE 1996 cAMP-response element modulator tau is a positive regulator of testis
angiotensin converting enzyme transcription. Proc Natl Acad Sci USA 93:1226212266[Abstract/Free Full Text]
-
Foulkes NS, Mellstrom B, Benusiglio E, Sassone-Corsi P 1992 Developmental switch of CREM function during spermatogenesis: from
antagonist to activator. Nature 355:8084[CrossRef][Medline]
-
Sassone-Corsi P 1998 Regulating the balance betwen
differentiation and apoptosis. J Mol Med 76:811817[CrossRef][Medline]
-
Osborne TF, LaMorte VJ 1998 Molecular aspects in feedback
regulation of gene expression by cholesterol in mammalian cells.
Methods 16:4248[CrossRef][Medline]
-
Shimano H, Horton J, Hammer RE, Shimomura I, Brown MS,
Goldstein JL 1996 Overproduction of cholesterol and fatty acids causes
massive liver enlargement in transgenic mice expressing truncated
SREBP-1a. J Clin Invest 98:15751584[Abstract/Free Full Text]
-
Athanikar JN, Osborne TF 1998 Specificity in cholesterol
regulation of gene expression by coevolution of sterol regulatory DNA
element and its binding protein. Proc Natl Acad Sci USA 95:49354940[Abstract/Free Full Text]
-
de Groot RP, Sassone-Corsi P 1993 Hormanal control of gene
expression: multiplicity and versatility of cyclic adenosine
3',5'-monophosphate-responsive nuclear regulators. Mol Endocrinol 7:145153[Medline]
-
Foulkes NS, Sassone-Corsi P 1992 More is better: activators
and repressors from the same gene. Cell 68:411414[Medline]
-
Walker WH, Girardet C, Habener JF 1996 Alternative exon
splicing controls translational switch from activator to repressor
isoforms of transcription factor CREB during spermatogenesis. J
Biol Chem 271:2014521050[Abstract/Free Full Text]
-
Sun Z, Means AR 1996 A role for cAMP-response element motifs
in transcriptional regulation of postmeiotic male germ cell-specific
genes. In: Hansson V, Levy FD, Taskén K (eds) Signal
Transduction in Testicular Cells. Springer-Verlag, Berlin, pp 3052
-
Sassone-Corsi P 1997 Transcriptional checkpoints determining
the fate of male germ cells. Cell 88:163166[Medline]
-
Blendy JA, Kaestner KH, Weinbauer GF, Nieschlag E, Schutz G 1996 Severe impairment of spermatogenesis in mice lacking the CREM
gene. Nature 380:162165[CrossRef][Medline]
-
Hecht NB 1990 Regulation of haploid expressed genes in
male germ cells. Reprod Fertil 88:679693
-
Bonny C, Cooker LA, Goldberg E 1998 Deoxyribonucleic
acid-protein interactions and expression of the human testis-specific
lactate dehydrogenase promoter: transcription factor Sp1 plays a major
role. Biol Reprod 58:754759[Abstract]
-
Aoyama Y, Funae Y, Noshiro M, Horiuchi T,Yoshida Y 1994 Occurrence of a P450 showing high homology to yeast lanosterol
14-demethylase (P45014DM) in the rat liver. Biochem Biophys Res Commun 201:13201326[CrossRef][Medline]
-
Sambrook J, Fritsch EF, Maniatis TM 1989 Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY
-
Strömstedt M, Kenney DS, Waterman MR, Paria BC, Conley
AJ, Dey SK 1996 Preimplantation mouse blastocysts fail to express CYP
genes required for estrogene biosynthesis. Mol Reprod Dev 43:428436[CrossRef][Medline]
-
Meistrich ML, Bruce WR, Clermont Y 1973 Cellular composition
of fractions of mouse testis following velocity sedimentation
separation. Exp Cell Res 79:213227[Medline]
-
Rainey WE, Bird IM, Mason JI 1994 The NCI-H295 cell-linea
pluripotent model for human adrenocortical studies. Mol Cell Endocrinol 100:4550[CrossRef][Medline]
-
Bird IM, Mason JI, Rainey WE 1994 Regulation of type-1
angiotensin-II receptor messenger-ribonucleic-acid expression in human
adrenocortical carcinoma H295 cells. Endocrinology 134:24682474[Abstract]
-
Deryckere F, Gannon F 1994 A one-hour minipreparation
technique for extraction of DNA-binding proteins from animal tissues.
Biotechniques 16:405[Medline]
-
Sanchez HB, Yieh L, Osborne TF 1995 Cooperation by sterol
regulatory element-binding protein and Sp1 in sterol regulation of low
density lipoprotein receptor gene. J Biol Chem 270:11611169[Abstract/Free Full Text]
-
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith
JA, Struhl K 1991 Current Protocols in Molecular Biology. John Wiley &
Sons Inc., New York
-
Shimomura I, Shimano H, Horton JD, Goldstein JL, Brown MS 1997 Differential expression of exons 1a and 1c in mRNAs for sterol
regulatory element binding protein-1 in human and mouse organs and
cultured cells. J Clin Invest 99:838845[Abstract/Free Full Text]
-
Shimano H, Horton JD, Shimomura I, Hammer RE, Brown MS,
Goldstein JL 1997 Isoform 1c of sterol regulatory element binding
protein is less active that isoform 1a in livers of transgenic mice and
culture cells. J Clin Invest 99:846854[Abstract/Free Full Text]
-
Chen R, Ingraham HA, Treacy MN, Albert VR, Wilson L, Rosenfeld
MG 1990 Autoregulation of pit-1 gene expression mediated by two
cis-active promoter elements. Nature 346:583586[CrossRef][Medline]
-
Nordeen SK, Green III PP, Fowlkes DM 1987 A rapid, sensitive,
and inexpensive assay for chloramphenicol acetyltransferase. DNA 6:173178[Medline]
-
Yoshida Y, Yamashita C, Noshiro M, Fukuda M, Aoyama Y 1996 Sterol 14-demethylase P450 activity expressed in rat gonads:
contribution to the formation of mammalian meiosis-activating sterol.
Biochem Biophys Res Commun 223:534538[CrossRef][Medline]