Cyclic Adenosine 3',5'-Monophosphate(cAMP)/cAMP-Responsive Element Modulator (CREM)-Dependent Regulation of Cholesterogenic Lanosterol 14{alpha}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Lanosterol 14{alpha}-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{tau}-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{tau} 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{tau}-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{tau} 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{tau}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}-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{alpha}-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. 1Go). 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{alpha}-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{alpha}-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 VII–XIV (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{alpha}-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{tau} (cAMP response element modulator-{tau}) 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{tau} in spermatids.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 2Go). 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. 3Go) 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. 3Go) 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. 4AGo). 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. 4BGo, lane 3) give a higher mol wt complex (supershift) in the presence of the crude anti-SREBP-1 (Fig. 4BGo, lane 2), and the binding is competed with excess of unlabeled CYP51-SRE1 probe (Fig. 4BGo, lane 5). Similarly, a SREBP protein complex is formed with CYP51-SRE1 using JEG-3 nuclear extracts (Fig. 4CGo). Anti-CREB (cAMP-response element binding protein) antibody doesn’t shift this complex (Fig. 4CGo, lane 1), while purified anti-SREBP-1 disrupts the complex (Fig. 4CGo, lane 5), and unlabeled CYP51-SRE1 competes the binding (Fig. 4CGo, 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 2–5). 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{tau} in Spermatids
In situ hybridization of mouse testis showed that CYP51 mRNA is abundantly present in the seminiferous epithelium (Fig. 5BGo). 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{alpha}-hydroxylase/17,20-lyase cytochrome P450 (CYP17, Fig. 5EGo). The intensity of the CYP51 signal varies greatly between different seminiferous tubule cross-sections (Fig. 5BGo), 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. 5DGo). In contrast, the signal is very strong in stages VII–VIII and is restricted to late round and early elongating spermatids immediately underneath the most mature elongated spermatids, which face the lumen (Fig. 5Go, 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. 5Go, 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{tau} (L. Monaco, P. Sassone-Corsi, unpublished data). This suggested a role for CREM{tau} 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. 6AGo) is not affected by the absence of CREM and is controlled by SREBP-1a as shown in Fig. 2Go. 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. 6AGo). This shows that haploid expression of CYP51 in mouse testis is CREM{tau}-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{tau} 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{tau} 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{tau} mechanism. Similar levels of squalene synthase mRNA are present in testis of normal, CREM-/+, and CREM-/- mice (Fig. 6BGo). Also, no CRE is found in the 5'-flank of the human squalene synthase gene (17).

CREM{tau} 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. 3Go). We wanted to evaluate whether purified CREM{tau} or CREM{tau} 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. 7AGo, lane 1). Both anti-CREB (Fig. 7AGo, lane 2) and anti-CREM (Fig. 7AGo, 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. 7BGo).



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. 8Go), 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. 8AGo, lane 5) and germ cells (Fig. 8BGo, lane 5), while the anti-CREB antibody doesn’t influence nuclear protein binding to CYP51-CRE2 (Fig. 8Go, 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{tau}-protein binds to CYP51-CRE1, CYP51-CRE2, and CYP51-CRE2new elements, yet with a lower affinity than to the consensus CRE TGACGTCA (Fig. 8CGo). 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{tau} to CYP51-CRE1 and CYP51-CRE2 elements and to consensus CRE from the somatostatin gene. CYP51-CRE2new was checked for binding of CREM{tau} 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. 9Go, lane 3), but no CYP51-SRE1 binding proteins can be detected in such extracts (Fig. 9Go, 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. 9Go, lane 2). This pattern is identical to that obtained with nuclear extracts from human somatic cells (Fig. 4Go). 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. 10Go). 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{tau} 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. 10Go, 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{tau} 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{tau} to mimic the physiology of haploid male germ cells. The human CYP51 promoter activity is stimulated 2.6-fold by CREM{tau} (lane 5) and 6.1-fold by the combined action of forskolin and CREM{tau} (lane 7), showing that CREM{tau} 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.



View larger version (9K):
[in this window]
[in a new window]
 
Figure 10. Human CYP51 Promoter (-334/+314) Can Stimulate the CAT Reporter Activity by cAMP-Dependent as Well as Sterol-Dependent Signaling Pathways

Transient transfections of JEG-3 cells were carried out using a CYP51 promoter cloned into pCAT-basic (lane 1, normalized to 1); lanes 2–7: cells were grown in different media and/or cotransfected with expression plasmids encoding SREBP-1a or CREM{tau} transcription factors. Sterol-dependent pathway—effects of: delipidated serum (lane 2), cotransfection by pCMVh SREBP 1a (lane 3), delipidated serum and cotransfection by pCMVh SREBP (lane 4). cAMP-dependent pathway—effects of: cotransfection by pSV CREM{tau} (lane 5), addition of forskolin (lane 6), cotransfection by pSVCREM{tau} and addition of forskolin (lane 7).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CYP51 encodes lanosterol 14{alpha}-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. 4AGo). 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. 10Go, strongly indicate that ATCACCTCAG is a functional SRE sequence.

Surprisingly, the ubiquitously expressed mammalian CYP51 mRNA encoding cholesterogenic lanosterol 14{alpha}-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{tau} (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{alpha}, CREMß, CREM{gamma}) in premeiotic germ cells to the activator forms (CREM{tau}, CREM{tau}1, CREM{tau}2) in postmeiotic germ cells (19). CREM{tau} 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{tau}-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{alpha}-demethylase belongs to this group of haploid-expressed transcripts induced by the transcription activator CREM{tau}. High expression of CYP51 in spermatids results from the appearance of shorter, germ cell-specific CYP51 transcripts (Fig. 9Go) (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{tau} during spermatogenesis. Increased levels of squalene synthase mRNA are observed before meiosis in pachytene spermatocytes (10) where the activator transcription factor CREM{tau} 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{tau}-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{tau}-dependent expression of CYP51 in spermatids. CYP51-CRE2 (Fig. 3Go) 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{tau} in germ cells of different species in vivo. In accordance to this, transfection assays (Fig. 10Go) show that CYP51 promoter containing only CYP51-CRE2 can efficiently mediate the CREM{tau}-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{tau} 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{tau}-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{tau}-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{tau} and apparently no coordinate-type regulation of cholesterogenic genes occurs in germ cells, it is likely that the cAMP/CREM{tau}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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' (331–351), antisense primer: 5'-GATGTCCTGGAGGAATGGT-3' (958–978). 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 50–100 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/Ham’s 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 manufacturer’s instructions (Bio-Rad Laboratories, Inc., Richmond, CA).

Expression and Purification of Recombinant CREM{tau} and SREBP-1a Proteins
Purification of bacterially expressed CREM{tau} protein was previously described (16). Recombinant SREBP-1a protein (amino acids 1–490) 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 manufacturer’s 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 301–407 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 {gamma}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 4–7 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{tau} 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 manufacturer’s 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 Z1–7225, J1–1380, 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Goldstein JL, Brown MS 1990 Regulation of the mevalonate pathway. Nature 343:425–430[CrossRef][Medline]
  2. 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 122:1122–1128[Abstract]
  3. Strömstedt M, Rozman D, Waterman MR 1996 The ubiquitously expressed human CYP51 encodes lanosterol 14{alpha}-demethylase, a cytochrome P450 whose expression is regulated by oxysterols. Arch Biochem Biophys 329:73–81[CrossRef][Medline]
  4. 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 74:252–254[Medline]
  5. 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 272:27107–27115[Abstract/Free Full Text]
  6. 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 95:1899–1902[Abstract/Free Full Text]
  7. 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 374:559–562[CrossRef][Medline]
  8. 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 58:1297–1302[Abstract]
  9. Byskov AG, Baltsen M, Andersen CY 1998 Meiosis-activating sterols: background, discovery and possible use. J Mol Med 76:818–823[CrossRef][Medline]
  10. Strömstedt M, Waterman MR, Haugen TB, Taskén K, Parvinen M, Rozman D 1998 Elevated expression of lanosterol 14{alpha}-demethylase (CYP51) and the synthesis of oocyte meiosis-activating sterols in postmeiotic germ cells of male rats. Endocrinology 139:2314–2321[Abstract/Free Full Text]
  11. Noshiro M, Aoyama Y, Kawamoto T, Gotoh O, Horiuchi T, Yoshida Y 1997 Structural and evolutionary studies on sterol 14{alpha}-demethylase P450 (CYP51), the most conserved monooxygenase. I. structural analyses of the gene and multiple sizes of mRNA. J Biochem 122:1114–1121[Abstract]
  12. Rozman D, Strömstedt, M, Tsui L-C, Scherer SW, Waterman MR 1996 Structure and mapping of the human lanosterol 14{alpha}-demethylase gene (CYP51) encoding the cytochrome P450 involved in cholesterol biosynthesis; comparison of exon/intron organization with other mammalian and fungal CYP genes. Genomics 38:371–381[CrossRef][Medline]
  13. Russell LD, Ettlin RA, Sinha Hikim AP, Clegg ED 1990 Histological and Histopathological Evaluation of the Testis. Choche River Press, Clearwater, FL
  14. Deleted in proof
  15. 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 380:159–162[CrossRef][Medline]
  16. 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 7:1502–1514[Abstract]
  17. 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 270:21958–21965[Abstract/Free Full Text]
  18. 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 93:12262–12266[Abstract/Free Full Text]
  19. Foulkes NS, Mellstrom B, Benusiglio E, Sassone-Corsi P 1992 Developmental switch of CREM function during spermatogenesis: from antagonist to activator. Nature 355:80–84[CrossRef][Medline]
  20. Sassone-Corsi P 1998 Regulating the balance betwen differentiation and apoptosis. J Mol Med 76:811–817[CrossRef][Medline]
  21. Osborne TF, LaMorte VJ 1998 Molecular aspects in feedback regulation of gene expression by cholesterol in mammalian cells. Methods 16:42–48[CrossRef][Medline]
  22. 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 98:1575–1584[Abstract/Free Full Text]
  23. 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 95:4935–4940[Abstract/Free Full Text]
  24. 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 7:145–153[Medline]
  25. Foulkes NS, Sassone-Corsi P 1992 More is better: activators and repressors from the same gene. Cell 68:411–414[Medline]
  26. 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 271:20145–21050[Abstract/Free Full Text]
  27. 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 30–52
  28. Sassone-Corsi P 1997 Transcriptional checkpoints determining the fate of male germ cells. Cell 88:163–166[Medline]
  29. Blendy JA, Kaestner KH, Weinbauer GF, Nieschlag E, Schutz G 1996 Severe impairment of spermatogenesis in mice lacking the CREM gene. Nature 380:162–165[CrossRef][Medline]
  30. Hecht NB 1990 Regulation of ’haploid expressed genes’ in male germ cells. Reprod Fertil 88:679–693
  31. 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 58:754–759[Abstract]
  32. 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 201:1320–1326[CrossRef][Medline]
  33. Sambrook J, Fritsch EF, Maniatis TM 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  34. 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 43:428–436[CrossRef][Medline]
  35. Meistrich ML, Bruce WR, Clermont Y 1973 Cellular composition of fractions of mouse testis following velocity sedimentation separation. Exp Cell Res 79:213–227[Medline]
  36. Rainey WE, Bird IM, Mason JI 1994 The NCI-H295 cell-line—a pluripotent model for human adrenocortical studies. Mol Cell Endocrinol 100:45–50[CrossRef][Medline]
  37. 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 134:2468–2474[Abstract]
  38. Deryckere F, Gannon F 1994 A one-hour minipreparation technique for extraction of DNA-binding proteins from animal tissues. Biotechniques 16:405[Medline]
  39. 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 270:1161–1169[Abstract/Free Full Text]
  40. 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
  41. 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 99:838–845[Abstract/Free Full Text]
  42. 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 99:846–854[Abstract/Free Full Text]
  43. 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 346:583–586[CrossRef][Medline]
  44. Nordeen SK, Green III PP, Fowlkes DM 1987 A rapid, sensitive, and inexpensive assay for chloramphenicol acetyltransferase. DNA 6:173–178[Medline]
  45. 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 223:534–538[CrossRef][Medline]