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Biology of Reproduction 65, 1452-1461 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

CCAAT/Enhancer Binding Protein ß Regulates Expression of the Cystatin-Related Epididymal Spermatogenic (Cres) Gene1

Nelson Hsiaa, and Gail A. Cornwall2,a

a Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

ABSTRACT

The CRES protein is a member of the cystatin superfamily of cysteine protease inhibitors with restricted expression in stage-specific germ cells, proximal caput epididymidis, and anterior pituitary gonadotroph cells. To elucidate the molecular mechanisms regulating the highly restricted expression of the cres gene, we have sequenced 1.6 kilobases of mouse cres 5' flanking sequence and performed studies to examine the cres gene promoter. Two putative CCAAT/enhancer binding protein (C/EBP) transcription factor binding motifs exist within the first 135 base pairs of cres promoter. Furthermore, our studies demonstrate that cres mRNA levels are dramatically reduced in the epididymides of C/EBPß-deficient mice. These data suggest that the C/EBP family of transcription factors, in particular C/EBPß, plays a role in the regulation of cres gene expression. In support of this finding, Northern blot analysis showed that C/EBPß is the predominant C/EBP family member expressed in the LßT2 gonadotroph cell line and the proximal caput epididymidis. Also, gel shift and supershift assays demonstrated that C/EBPß protein in nuclear extracts from LßT2 gonadotroph cells and epididymal cells bound to the two C/EBP sites in the cres promoter. Finally, to test the in vivo function of the C/EBP sites in cres gene expression, transfection studies were performed in LßT2 gonadotroph cells and two heterologous cell systems. These experiments showed a significant reduction of cres transactivation when either C/EBP sites were mutated, and no transC/EBP activation of the cres promoter when both C/EBP sites were mutated. Taken together, these studies demonstrate that the C/EBPß transcription factor is necessary for high levels of cres gene expression in the proximal caput epididymidis and anterior pituitary gonadotroph cells.

anterior pituitary, epididymis, gene regulation, male reproductive tract, sperm maturation

INTRODUCTION

It is well known that spermatozoa exiting the testis are nonfunctional gametes and become fully mature only after exposure to proteins synthesized and secreted by the epididymis in a highly region-dependent manner. The regional expression of genes and subsequent regionalized secretion of epididymal proteins results in spermatozoa encountering proteins in a precise order as they migrate through the epididymal tubule, which is believed to be critical for the proper maturation of motile and fertile spermatozoa. During a search to identify genes expressed in the most proximal region of the epididymis and thus likely to be involved in the initiation of sperm maturation, we identified a unique gene we termed cres [1].

The cystatin-related epididymal spermatogenic (cres) gene represents a new subgroup within the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors [2]. Unlike the ubiquitous expression of the cystatin genes, cres gene expression is restricted to the proximal caput epididymal region, stage-specific germ cells in the testis, and the anterior pituitary gonadotroph cells, suggesting specific roles in reproduction [1, 3, 4]. Furthermore, in the epididymis, the cres gene is dependent upon unknown testicular factors for expression [1], whereas in the anterior pituitary gonadotroph cells cres gene expression is regulated by GnRH (unpublished observations).

In addition to cres, many other genes exhibit highly regionalized expression in the epididymis, including gamma glutamyl transpeptidase, glutathione peroxidase, proenkephalin, PEA3, and bone morphogenetic proteins 7 and 8a in the proximal caput region; procathepsin L, CRISP1, and E-RABP in the distal caput region; CD52, nerve growth factor, and beta hexosaminidase in the corpus region; and CE10 in the cauda region [5–8]. To date, however, little is known regarding the transcriptional mechanisms controlling the tissue-, region-, and cell-specific expression of genes in the epididymis. Similarly, the selective expression of genes, including that of the gonadotropins LH and FSH, in the gonadotroph cells of the anterior pituitary gland is critical for normal gonadotroph function. However, the molecular mechanisms important for high levels of gene expression in the gonadotroph cells are also not well understood. To begin to elucidate the molecular mechanisms responsible for basal as well as tissue-, region-, and cell-specific expression of the cres gene, studies of the cres gene promoter were initiated.

An analysis of the mouse cres gene 5'-flanking sequences revealed the presence of two consensus CCAAT/enhancer-binding protein (C/EBP) binding sites within the first 135 base pairs (bp) of the cres promoter. C/EBP is a family of basic region/leucine zipper transcription factors implicated in the regulation of a variety of genes involved in energy metabolism and cell differentiation pathways. Currently, there are several C/EBP family members, including {alpha}, ß, {gamma}, {delta}, {epsilon}, and {zeta} forms. C/EBP{alpha}, ß, and {delta} have been found in tissues such as liver, adipose tissue, intestine, lung, and cells of the inflammatory system, whereas C/EBP{epsilon} is restricted to myeloid and lymphoid lineages [9]. Recently, C/EBP family members have been implicated in several processes in the male and female reproductive tracts. In females, C/EBPß is critical for ovarian follicular development specifically as a downstream target of LH receptor signaling [10]. Similarly, in the male, studies suggest that LH-dependent gene regulation in the Leydig cells [11] and FSH-dependent gene regulation in Sertoli cells [12] may be mediated by C/EBPß. In addition, C/EBP{delta} expression in epididymis and prostate is up-regulated following castration suggesting androgen-regulated functions in these tissues [13]. Because the cres gene, as well as several other genes expressed in the initial segment/proximal caput region of the epididymis, including mEP17 [14] and GGT [15], possess consensus C/EBP sites in their proximal promoters, C/EBP family members may be involved in the regulation of gene expression in the epididymis. In addition, previous studies have also suggested a role for C/EBP family members in gene regulation in the pituitary gland [16]. Therefore, the present experiments were designed to determine whether the C/EBPß transcription factor is important for high levels of cres gene expression in the proximal caput epididymidis and the anterior pituitary gonadotroph cells.

MATERIALS AND METHODS

Animals

Retired breeder and 28-day-old male CD-1 strain mice were obtained from Charles River (Wilmington, MA). Mice were housed under a constant 12L:12D cycle and allowed free access to food and water.

Plasmids

A firefly luciferase reporter construct containing 135 bp of cres promoter sequence was generated by polymerase chain reaction amplification from a 1.6-kilobase (kb) cres promoter fragment and insertion into the pGL3-Basic vector (Promega, Madison, WI). Sequences of cres primers used are as follows with restriction sites underlined: 5'-GGCCAGAAGATGAAGAG-3' and 5'-TTGTGGTTCTTCTGG-3'. The 135-bp PCR product was digested with KpnI and SmaI and ligated into the pGL3-Basic linearized with the same restriction enzymes. Mutant cres reporter constructs were prepared using the wild-type 135 bp cres-pGL3-Basic construct. We introduced three bp substitutions that are known to disrupt C/EBP binding into the C/EBP sites at -88/-78 and -48/-40 bp using the Quikchange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Double-stranded sequence analysis was performed with {alpha}-35S dATP (New England Nuclear, Boston, MA) and the Sequenase II kit (Amersham, Cleveland, OH) to verify the presence of mutations. The pRL-TK Renilla luciferase control reporter vector (Promega) was used for normalization of luciferase data. The pMEX-C/EBPß expression vector was a generous gift from Dr. Simon Williams, Texas Tech University Health Sciences Center (TTUHSC), Lubbock, Texas. Plasmids for transfections were prepared using the double-banded cesium chloride method as described [17].

RNA Isolation and Northern Blot Analysis

Total RNA was isolated from mouse tissues and LTß2 gonadotroph cells (a gift from Dr. Pam Mellon, University of California at San Diego, La Jolla, CA) with the use of Trizol reagent (Gibco BRL, Grand Island, NY) in accordance with the manufacturer's protocol. For the preparation of RNA from C/EBPß and C/EBP{delta} heterozygous and homozygous null mice, testes and epididymides were pooled from four to six mice (age 60–120 days; kindly provided by Dr. Peter Johnson, National Cancer Institute, Frederick, MD). The RNA samples were heated at 95°C for 2 min and then loaded onto a gel of 1% agarose, 2% formaldehyde, and 1x borate gel, and electrophoresed. The gels were washed extensively in water to remove formaldehyde before transferring to a nylon membrane (Nytran, Schleicher & Schuell, Keene, NH) by vacuum blotting (Appligene, Illkirch, France) for 3 h in the presence of 10x saline-sodium citrate (SSC). The membranes were then washed for 5 min in 5x SSC before UV cross-linking (Stratalinker, Stratagene). The membranes were prehybridized for 2 h at 42°C in hybridization buffer containing 50% formamide, 5x SSC, 0.2 mg/ml salmon sperm DNA, 0.4 mg/ml yeast RNA, 50 µg/ml BSA, 0.1% SDS, and 12.5 mM sodium phosphate buffer pH 6.6, followed by hybridization overnight at 42°C in the presence of 3 x 106 cpm probe/ml hybridization buffer. The cDNA probes were prepared using a random prime labeling method (Prime-It II, Stratagene; C/EBP {alpha}, ß, {delta}, and {epsilon} cDNAs were a generous gift of Dr. Simon Williams, and mEP17 cDNA was a generous gift of Dr. Marie-Claire Orgebin-Crist, Vanderbilt University, Nashville, Tennessee). After hybridization, the blots were rinsed in 2x SSC at room temperature followed by washes in 2x SSC and 1% SDS at 42°C for 30 min, and then at 65°C for 30 min before exposure to film. To verify equal transfer of RNA onto the blot, the cDNA probes were removed from each blot by stripping twice at 55°C for 30 min in 0.1x SSC and 1% SDS that had been preheated to 100°C. The stripped blots were then reprobed under the same conditions described with a cDNA probe to 18S rRNA.

To quantitate the amount of cres, B-myc, cystatin c, and mEP17 mRNAs in the epididymides of C/EBPß heterozygous and homozygous null mice, autoradiograms were scanned using a computer-assisted image analysis system (Fluorchem 8000, Alpha Innotech Corp., San Leandro, CA). The integrated areas obtained for each mRNA were then normalized to the areas obtained for the 18S ribosomal signal. The normalized values for mRNAs expressed in homozygous null tissues were then compared with those in heterozygous tissues, and the percent increase or decrease was reported.

In Situ Hybridization

Paraformaldehyde-fixed mouse epididymides were embedded in paraffin wax, and tissue sections of 6–8 µm were cut and mounted onto Superfrost (Fisher Scientific) glass slides by the TTUHSC Electron Microscopy Core Facility. Tissue sections were hybridized with 100 µl of hybridization solution containing 50% deionized formamide, 300 mM sodium chloride, 20 mM Tris pH 8, 5 mM EDTA pH 8, 1x Denhardt (0.02% Ficoll, 0.02% polyvinylpyrrolindone, 0.02% BSA), 10% dextran sulfate, and 10 mM dithiothreitol (DTT) and approximately 1 x 106 cpm of an 35S-labeled antisense C/EBPß RNA probe representing the 3' untranslated region of the C/EBPß mRNA. Hybridization was carried out overnight at 55°C in a humidified chamber. Following hybridization, the sections were washed twice in 2x SSC, 10 mM ß-mercaptoethanol, and 1 mM EDTA for 10 min at room temperature. The slides were then washed in 500 mM NaCl, 10 mM Tris-Cl pH 8 containing 20 µg/ml RNase A, for 30 min at room temperature, followed by two 10-min washes at room temperature in 2x SSC, 10 mM ß-mercaptoethanol, 1 mM EDTA. Slides were washed for 2 h in 0.1x SSC, 10 mM ß-mercaptoethanol, 1 mM EDTA at 55°C with a change after 5 min, 20 min, and 95 min, followed by two washes in 0.5x SSC at room temperature. The slides were then dehydrated through ethanol solutions (50%, 70%, 90%, 100%, and 100%) containing 0.3 M NH4Ac. The slides were dipped in Kodak NTB-2 emulsion, dried, and allowed to expose for 14 days. The exposed slides were developed, fixed, and stained with toluidine blue, and coverslips were applied with Permount.

Preparation of Nuclear Extracts and Western Blot Analysis

Nuclear extracts were prepared as previously described [18]. Briefly, 1 x 106 LßT2 gonadotroph cells were washed in 10 ml of cold PBS, and pelleted cells were resuspended in 400 µl of ice-cold low-salt buffer A (10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF). The cells were allowed to swell for 15 min on ice and then 25 µl of 10% NP-40 was added. The cells were vigorously vortexed, and the nuclei were centrifuged for 30 sec at 13 800 x g in a microfuge. Nuclear pellets were resuspended in 50 µl of high-salt buffer C (20 mM Hepes pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF) and gently rocked at 4°C for 20 min. The nuclear lysate was centrifuged at 13 800 x g for 5 min at 4°C, and protein concentration of the supernatant was determined using the bicinchoninic acid assay (Pierce, Rockford, IL). Extracts were immediately used for electrophoretic mobility shift assays (EMSAs) or aliquoted and stored at -80°C until required.

For the preparation of epididymal nuclear extracts, epididymal tissue from 28-day-old mice was used to avoid the contamination of sperm proteins in the nuclear extract. Spermatogenesis is actively carried out in these mice; however, at 28 days of age spermatozoa have not yet entered the epididymis [19]. The cres gene is expressed in the epididymides of 28-day mice, albeit at slightly lower levels than in more mature mice (unpublished observations). The proximal caput epididymidis (Fig. 2A, region 1) of 5 mice were dissected and immediately frozen and stored at -80°C. Frozen epididymides were thawed on ice and homogenized in ice-cold SSHB buffer (0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 15 mM Tris-HCl pH 7.5, 0.5 mM spermidine, 0.15 mM spermine, 2 mM EDTA, 0.5 mM EGTA, and 14 mM 2-mercaptoethanol) using a polytron homogenizer. Nuclei were filtered through a 70-micron mesh to separate the nuclei from connective tissue, and the homogenate was centrifuged at 1000 x g. The nuclear pellet was washed several times in SSHB/0.1% Triton X-100, and clean nuclei were divided into 1 x 106 nuclei aliquots. These nuclear pellets were resuspended in 50 µl of high-salt buffer C and processed as described above for LßT2 cell nuclear extracts.



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  		FIG. 2. Expression of C/EBP family members in the LßT2 gonadotroph cell line and regions of the mouse epididymis. Total RNA was isolated from LßT2 gonadotroph cells and the five epididymal regions as depicted in A: 1, proximal caput; 2, mid caput; 3, distal caput; 4, corpus; 5, cauda epididymidis. B) Northern blot analysis of 10 µg of total RNA from the LßT2 gonadotroph cells and five epididymal regions probed with C/EBPß cDNA. The blot was stripped and reprobed with C/EBP{alpha} and C/EBP{delta} cDNAs followed by 18S to confirm equal loading of RNA. Autoradiographic exposure times for the blots were C/EBP{alpha}, 4 days; C/EBPß, 4 days; and C/EBP{delta}, 2 wk

Western blot analysis was carried out to examine C/EBPß protein levels in transfected heterologous cells. Briefly, equal amounts of cell lysate were loaded and separated on a 15% SDS-polyacrylamide gel. After electrophoresis, proteins were blotted onto Protran membranes (Schleicher & Schuell) in a solution containing 0.1% SDS, 20% methanol, 400 mM glycine, and 50 mM Tris-HCl pH 8.3 at 4°C for 4 h at a constant current of 200 mA. The blot was blocked with 5% milk, 1x PBS, and 0.2% Tween-20 for 1 h at room temperature. The blot was then incubated for 2 h at 4°C with primary C/EBPß rabbit antiserum (obtained from Dr. Simon Williams) diluted 1:4000 in 2% milk, 1x PBS, and 0.2% Tween-20, and then incubated for 30 min at room temperature with a goat anti-rabbit secondary antibody (BioSource International, Camarillo, CA) diluted 1:40 000 in 2% milk, 1x PBS, and 0.2% Tween-20. The Supersignal West Pico Chemiluminescent substrate kit (Pierce) was used for antigen-antibody complex detection.

Electrophoretic Mobility Shift Assays

Double-stranded oligonucleotide probes 19–21 bases in length were end-labeled with [{gamma}-32P]ATP (New England Nuclear) with T4 polynucleotide kinase (Promega). Briefly, 10 pmol of sense strand oligonucleotide was incubated for 1 h at 37°C in a T4 polynucleotide kinase reaction. Then, 30 pmol of antisense oligonucleotide was added, and NaCl was added to a final concentration of 100 mM. This reaction was incubated for 5 min at 95°C and then slowly cooled to room temperature. The reaction was centrifuged through a G-25 spin column (Roche Molecular Biochemicals) to remove free nucleotide and unhybridized oligonucleotides. Labeled probes (50 000 cpm total) were added to a 25-µl binding reaction containing 5 µl of 5x binding buffer (20% [w/v] Ficoll, 50 mM Hepes pH 7.9, 50 mM DTT, and 5 mM EDTA pH 8.0), 1 µg of poly(dI-dC), and 3 µl of a 50-µl in vitro-transcribed and -translated recombinant C/EBPß protein reaction (a gift from Dr. Elmus Beale, TTUHSC, Lubbock, TX), 10 µg of LßT2 nuclear extracts, or 10 µg of proximal caput epididymal nuclear extracts, and incubated for 30 min at 4°C. Competition assays were performed by adding a 200 molar excess of unlabeled oligonucleotides for 30 min at 4°C prior to addition of labeled probe. In supershift experiments, 2 µg of C/EBP{alpha}, -ß, or -{delta} affinity-purified antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) or 1 µl C/EBPß rabbit-antiserum was added to the binding reaction for an additional incubation of 30 min at 4°C. The protein/DNA complexes were separated from free probe by electrophoresis in a 5% nondenaturing polyacrylamide gel in 0.5x TBE buffer (50 mM Tris borate, 50 mM boric acid, 1 mM EDTA), and dried gels were exposed for autoradiography. The oligonucleotides used in the gel shift assay were as follows, with the core binding site underlined and bp mutations in lowercase (WT, wild-type; M, mutant; see Fig. 1 for the location of C/EBP1 and C/EBP2 sites): C/EBP1WT, 5'-CAGGACTGCC-3' and 3'-GTCCTGACGG-5'; C/EBP1M, 5'-CAGGACTGCC-3' and 3'-GTCCTGACGG-5'; C/EBP2WT, 5'-TGCTAAAGCG-3' and 3'-ACGATTTCGC-5'; and C/EBP2M, 5'-TGCTAAAGCG-3' and 3'-ACGATTTCGC-5'.



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  		FIG. 1. Sequence of the cres gene promoter and identification of consensus DNA binding sites. Mouse cres 5'-flanking sequences were examined for transcription factor binding sites using the MatInspector program [39]. Consensus motifs are boxed and labeled. The location of the C/EBP sites and mutations created to abolish C/EBP protein binding are shown below the cres promoter sequence. The sequences of the wild-type oligonucleotides used for EMSAs (wt EMSA oligos) are also noted

Cell Culture and Transfection

HeLa and human hepatoma HepG2 cells (American Type Culture Collection, Manassas, VA) and LßT2 gonadotroph cells were cultured in Dulbecco modified Eagle medium (DMEM) high glucose supplemented with 5% fetal calf serum, 5% calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 5% CO2 and 37°C in a humidified incubator. Media and antibiotics were purchased from Gibco BRL and serum was purchased from Hyclone (Logan, UT). HepG2 and HeLa cells were plated at 80 000 cells/well in 24-well Corning plates (Corning, Acton, MA) the day prior to transfection. Cells were washed in DMEM without serum and then transfected with Cytofectene (Bio-Rad, Hercules, CA) plus plasmid DNA as described by the manufacturer's protocol. Cells were transfected with 200 ng of cres-pGL3-Basic vector and 0, 100, 150, or 300 ng of the pMEX-C/EBPß expression vector. The total amount of DNA transfected in each culture well was made up to 500 ng with empty pMEX expression vector. Because C/EBPß is known to activate the pRL-TK Renilla luciferase control vector, it was not included in the heterologous transfection experiments in which the dose-response of C/EBPß on cres promoter activity was examined. The production of C/EBPß protein was confirmed, however, by Western blot analysis of each cell transfection. After 48 h, cells were harvested by scraping them in 100 µl of reporter lysis buffer (Promega, Madison, WI) followed by a single freeze/thaw cycle. Heterologous cell lysates were then assayed for firefly luciferase on a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA) using substrates in the Luciferase Assay Kit (Promega). Cres pGL3-Basic vector luciferase activity was normalized to that of empty pGL3-Basic vector in the absence of pMEX-C/EBPß expression vector, and the mean ± SEM of four independent experiments is presented.

Transfection of LßT2 cells (1 x 106 cells/well in a 6-well plate) utilized the Transfast (Promega) protocol as described by the manufacturer. LßT2 cells were transfected with 2 µg of the cres-pGL3-Basic vector and 50 ng of pRL-TK Renilla luciferase vector as an internal control for transfection efficiency. After 48 h, cells were harvested by scraping them in 100 µl reporter lysis buffer followed by a single freeze/thaw cycle. Firefly and Renilla luciferase activity from LßT2 cell lysates were measured using buffers and substrates provided by the Dual-Luciferase Kit (Promega). Data are presented as the ratio of firefly luciferase to Renilla luciferase (L/R) and represent mean ± SEM of four independent experiments.

RESULTS

Identification of C/EBP Response Elements Within the Cres Gene Promoter

Previous studies have characterized the genomic structure and the promoter sequences of the mouse cres gene [2]. An analysis of potential transcription factor binding sites within the proximal promoter revealed the presence of two estrogen response elements (EREs) and one steroidogenic factor-1 (SF-1) element (Fig. 1). Two binding sites that exhibit similarity to the consensus site (ATTGCGCAAT) for the CCAAT/enhancer binding protein family of transcription factors were also identified within a 135-bp region of cres proximal promoter (Fig. 1). In general, naturally occurring C/EBP binding sites do not exhibit the exact consensus site; rather, they consist of a well-conserved half site paired with a more divergent site that usually has at least two base pairs of similarity to the consensus, typically the G and A at nucleotides 1 and 4, respectively, of the GCAAT consensus [20]. In the cres gene promoter a perfect consensus half site GCAAT is present at -78 to -88 bp paired with a half site containing a conserved A at nucleotide position 4 of the consensus (C/EBP site 2). An additional potential C/EBP binding site was also identified at -39 to -48 bp (C/EBP site 1) consisting of two divergent half-sites; one with the G and A nucleotides conserved at positions 1 and 4, respectively, of the consensus sequence and one half-site with a conserved A at position 4.

Expression of C/EBP mRNAs in a Gonadotroph Cell Line and Regions of the Epididymis

The presence of two C/EBP motifs in the proximal promoter suggests that C/EBP proteins may play a role in the regulation of the cres gene. To address this we first looked for the presence of C/EBP family members in cells or tissues expressing the cres gene, including the anterior pituitary gonadotroph cells and the proximal caput epididymidis. Northern blot analyses showed expression of the C/EBP{alpha}, -ß, and -{delta} genes in the LßT2 gonadotroph cell line, a differentiated gonadotroph cell line characterized by its expression of both the {alpha}-subunit gene and LHß-subunit gene that determines hormone specificity [21] (Fig. 2B). Although we cannot rule out that the specific activities of the cDNA probes may be different, autoradiographic exposure times for the blots suggest that in the gonadotroph cells, the mRNA expression levels are highest for C/EBPß, intermediate for C/EBP{alpha}, and lowest for C/EBP{delta}.

The mouse epididymis can be sectioned into five general regions that are depicted in Figure 2A, and include the caput (regions 1–3), corpus (region 4), and cauda (region 5). We have previously demonstrated that the cres mRNA is exclusively expressed in the epithelial cells of the proximal caput epididymis (region 1) [1]. Northern blot analysis of C/EBP family members in the epididymis showed a regionalized pattern of mRNA expression (Fig. 2B). Specifically, C/EBPß was the predominant C/EBP family member expressed and was primarily found in the same region as the cres gene, the proximal caput epididymidis (region 1), with decreasing levels of expression in distal epididymal regions. C/EBP{alpha} was also expressed in the proximal caput epididymal region, although at much lower levels. C/EBP{delta} mRNA was expressed weakly throughout all regions of the epididymis with highest expression in the mid caput, region 2. Taken together, these data show that, although all three C/EBP family members are expressed, C/EBPß is the predominant family member in the gonadotroph cells and epididymis. Expression of C/EBP{epsilon} mRNA was also examined in the LßT2 gonadotroph cells and the epididymis, but was not detectable by Northern blot analysis (data not shown). Because the cres gene is also expressed in testicular germ cells [3], expression of the C/EBP{alpha}, ß, and {delta} genes was examined in whole testis mRNA by Northern blot analysis. However, relative to expression in the epididymis, no detectable mRNA was observed for the three C/EBP family members (data not shown).

Expression of C/EBPß mRNA in the Epithelial Cells of the Mouse Epididymis

To identify the one or more cell populations expressing the C/EBPß gene in the epididymis, in situ hybridization was performed. As shown in Figure 3, A and B, C/EBPß mRNA was detected in the epididymis with highest levels of expression in the proximal caput (region 1) with less expression in the mid caput (region 2) and throughout the remaining epididymal regions (not shown), thereby supporting our Northern blot studies. Only background levels of silver grains were detected in the epididymides from C/EBPß homozygous null mice, thus confirming the specificity of the signal for the C/EBPß mRNA (Fig. 3, E and F). Although we cannot eliminate the possibility that other cell types in the epididymis also express C/EBPß mRNA, examination of a higher magnification of the proximal caput region showed silver grains over the principal epithelial cells (Fig. 3, C and D). Therefore, these studies indicate that the C/EBPß gene is expressed in the same cell population as the cres gene.



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  		FIG. 3. In situ hybridization of the antisense C/EBPß RNA to the mouse epididymis. Longitudinal sections of the mouse epididymis from A and B) C/EBPß heterozygous (+/-) and E and F) C/EBPß homozygous null (-/-) mice were hybridized with an 35S-labeled antisense riboprobe to the C/EBPß cDNA and photographed under A and E) brightfield and B and F) darkfield illumination. Bar = 320 µm. C, D) Higher magnification of an epididymal section from a C/EBPß heterozygous (+/-) mouse probed with the C/EBPß antisense riboprobe. Bar = 91 µm. C/EBPß mRNA appears as dark grains under brightfield illumination and white grains under darkfield illumination. 1, Proximal caput; 2, mid caput epididymal region

Cres Gene Expression in the Epididymides of C/EBPß Heterozygous and Homozygous Null Mice

The existence of C/EBP transcription factor motifs in the cres promoter and the expression of C/EBP family members in cells and tissues expressing the cres gene indicate the appropriate elements are present for C/EBP activation of the cres promoter. To assess the biological significance of C/EBP proteins in the regulation of cres gene expression in vivo, we examined the expression levels of cres mRNA in the epididymides of C/EBP gene knockout mice. As shown in Figure 4, the cres gene showed a dramatic 71% decrease in expression in the epididymides of C/EBPß homozygous null (-/-) mice compared with that in the epididymides of C/EBPß heterozygous mice (+/-). Cres expression in epididymides of C/EBPß heterozygous (+/-) mice, however, was not different from that in C/EBPß wild-type (+/+) mice (data not shown). Examination of cres expression in the testes of C/EBPß heterozygous and homozygous null mice showed no difference, suggesting that C/EBPß may not be involved in cres gene regulation in testicular germ cells. In addition to examining C/EBPß knockout mice, we also examined cres mRNA levels in the epididymides of C/EBP{delta} knockout mice [22]. However, no difference in cres mRNA levels was observed between the epididymides and testes of heterozygous and C/EBP{delta} homozygous null mice (data not shown). Finally, it was not possible to analyze tissues from C/EBP{alpha}-deficient mice because these mice die perinatally due to hypoglycemia and other complications [23]. These studies suggest that C/EBPß is necessary for high levels of cres expression in the epididymis.



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  		FIG. 4. Expression of the cres gene in epididymides and testes of C/EBPß heterozygous and homozygous null mice. Ten micrograms of total RNA from whole epididymides and testes of C/EBPß heterozygous (+/-) and homozygous null (-/-) mice were examined for cres, C-EBP, B-myc, cystatin c, and mEP17 expression by Northern blot analysis. The blots were stripped and reprobed with the cDNA for 18S rRNA to confirm equal loading of RNA. This experiment has been repeated two to three times using two different preparations of RNA from heterozygous and C/EBPß homozygous null deficient tissues. Representative Northern blots are shown. Compared with mRNA levels in the heterozygous control epididymides, cres mRNA levels were decreased by 64%, 78%, and 68% in homozygous null epididymides; B-myc mRNA levels were decreased by 37% and increased by 15% and 11% in homozygous null epididymides; mEP17 mRNA levels were decreased by 15% and increased by 22% in homozygous null epididymides. Cystatin c mRNA levels were decreased by 1.7% in homozygous null epididymides

We also examined the expression of several other genes in the epididymides and testes of C/EBPß-deficient mice to determine whether the loss of C/EBPß had a broad effect on proximal caput-specific gene expression or was limited to selected genes. Expression of B-myc, which is predominantly expressed in the proximal caput epididymal region with less expression in distal epididymal regions [23], cystatin c, ubiquitously expressed in all epididymal regions [1], and mEP17, exclusively expressed in the proximal caput region [14], showed only minor differences in mRNA levels between C/EBPß heterozygous and homozygous null epididymides that did not appear to correlate with the loss of the C/EBPß gene (Fig. 4, legend). These data suggest the specific involvement of the transcription factor C/EBPß in the regulation of cres gene expression in the epididymis.

Recombinant C/EBPß Protein Binds to Cres C/EBP Oligonucleotides

To determine whether C/EBPß protein is able to form specific DNA/protein complexes with cres C/EBP sequences, EMSAs were performed (Fig. 5). Recombinant C/EBPß protein formed complexes with wild-type labeled cres C/EBP site 1 (1WT) and 2 (2WT) oligonucleotides (see Fig. 1 for oligonucleotide sequence), as shown in lanes 1–3 and 7–9 (lower arrow, Fig. 5). However, when three base pair mutations known to disrupt C/EBP binding were introduced into the cres C/EBP site 1 (1M) and 2 (2M) sequences, recombinant C/EBPß was unable to bind and complexes were not detected (lanes 4–6 and 10–12). To confirm the presence of C/EBPß protein in the DNA/protein complex, supershift assays were conducted in which C/EBPß antiserum (ß in Fig. 5) or normal rabbit serum (S in Fig. 5) was added after incubation of recombinant C/EBPß protein and oligonucleotide probe. A supershifted complex (upper arrow) observed in samples incubated with C/EBPß antibody (lanes 3 and 9) confirmed the presence of C/EBPß protein in the DNA/protein complexes (Fig. 5). Incubation of the binding reaction with normal rabbit antiserum did not result in a supershifted complex, demonstrating the specificity of the antibody for the recombinant C/EBPß protein (lanes 2 and 8). These data demonstrate that C/EBPß protein specifically binds to the cres C/EBP site 1 and 2 sequences.



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  		FIG. 5. EMSA with recombinant C/EBPß protein and cres C/EBP oligonucleotides. In vitro transcribed and translated C/EBPß protein was incubated with 32P-labeled oligonucleotide probes to wild-type (1WT, 2WT) and mutant (1M, 2M) cres C/EBP sites 1 and 2 (see Fig. 1 for sequence). Lower arrow indicates shifted complexes observed with wild-type but not mutant oligonucleotides. Addition of C/EBPß antiserum (ß) to DNA/protein complexes resulted in a supershifted complex (upper arrow, lanes 3 and 9), whereas addition of normal rabbit serum (S) did not result in a supershifted complex, indicating specificity of the antibody for C/EBPß protein. (-) Indicates no antibody added

Endogenous C/EBPß Protein Binds to Cres C/EBP Oligonucleotides

Although we have shown that recombinant C/EBPß protein binds the cres gene promoter, the binding capacity of endogenous C/EBPß protein in cres-expressing tissues required further investigation. To address this, EMSAs were carried out using nuclear extracts prepared from LßT2 gonadotroph cells and proximal caput epididymidis and 32P-labeled oligonucleotides representing cres C/EBP sites 1 and 2. As shown in Figure 6, A and B, incubation of nuclear extracts from the LßT2 gonadotroph cells and proximal caput epididymidis, respectively, resulted in the formation of DNA/protein complexes with cres C/EBP site 1 (1WT; lane 1) and 2 (2WT; lane 7) sequences as indicated by the lower two arrows. The addition of 200x molar excess of unlabeled wild-type but not mutant cres C/EBP site 1 and 2 oligonucleotides resulted in a decrease in the formation of DNA/protein complexes, demonstrating a specific competition by unlabeled wild-type oligonucleotide (lanes 2 and 8) and a lack of competition by the mutant oligonucleotide (lanes 3 and 9). Supershift experiments using specific antibodies that recognize C/EBP {alpha}, ß, or {delta} proteins were next carried out to identify the C/EBP family members in the nuclear extracts that interact with the cres C/EBP site 1 and 2 sequences. As indicated by the upper arrow in Figure 6A, both cres C/EBP 1WT and 2WT oligonucleotides specifically interact with C/EBPß protein present in LßT2 nuclear extracts as indicated by the presence of a supershifted complex in the presence of C/EBPß antibody (lanes 5 and 11). The lack of supershifted complexes in the presence of C/EBP{alpha} and -{delta} antibodies indicate the absence of binding of these C/EBP family members (Fig. 6A, lanes 4, 6, 10, and 12). Similarly, as indicated by the presence of specific supershifted complexes (Fig. 6B, lanes 5 and 11), C/EBPß protein is the predominant C/EBP family member in proximal caput epididymal nuclear extracts that binds to cres C/EBP sites 1 (1WT) and 2 (2WT) oligonucleotides. The occurrence of a minor supershifted complex in the presence of C/EBP{alpha} antibody (Fig. 6B, lanes 4 and 10) suggests that in the epididymis, a slight amount of C/EBP{alpha} protein may also bind to the cres C/EBP sites. These data suggest that endogenous C/EBPß in the gonadotroph cells of the pituitary and the proximal caput epididymidis is able to form DNA/protein complexes with C/EBP sites in the cres gene promoter.



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  		FIG. 6. EMSAs with LßT2 gonadotroph cell and proximal caput epididymal nuclear extracts and cres C/EBP oligonucleotides. Nuclear extracts prepared from A) LßT2 gonadotroph cells and B) proximal caput epididymidis were incubated with 32P-labeled oligonucleotides representing cres C/EBP sites 1 (1WT) and 2 (2WT) and examined on nondenaturing acrylamide gels. The shifted complexes are indicated by the two lower arrows (lanes 1 and 7). The addition of 200x molar excess of unlabeled wild-type (200x 1, 2WT) (lanes 2 and 8) but not mutant (200x 1, 2M) (lanes 3 and 9) cres C/EBP site 1 and 2 oligonucleotides resulted in a decrease in the shifted complexes indicating competition by wild-type but not mutant oligonucleotides. When antibodies against different C/EBP family members were added to the DNA/protein complexes, supershifted complexes (upper arrow) were detected primarily with C/EBPß antibody (lanes 5 and 11). A minor supershifted complex was also observed with C/EBP{alpha} antibody and proximal caput epididymal extracts. Ten micrograms of nuclear extract were used in each lane

Activation of the Cres Promoter in LßT2 Gonadotroph Cells

Studies were next carried out to determine whether the C/EBPß transcription factor is functionally important for activation of the cres gene promoter. The ability of endogenous C/EBPß protein in LßT2 cells to transactivate the cres promoter was determined by transfection of LßT2 cells with wild-type or mutated cres-pGL3-Basic vectors (Fig. 7A) and pRL-TK Renilla luciferase vector to control for transfection efficiency. Transfection of the 135-bp fragment of cres promoter (135 bp) into the LßT2 cells showed approximately a 4-fold increase in activation over empty reporter vector (pGL3-Basic; Fig. 7B). Mutation of cres C/EBP site 1 (1M) to a nonconsensus C/EBP site resulted in a greater than 50% reduction in activation of the luciferase reporter gene, whereas mutation of cres C/EBP site 2 (2M) resulted in an even more profound decrease in reporter gene activation to levels similar to that of empty vector. Finally, transfection of LßT2 cells with a construct in which both cres C/EBP sites were mutated (DM) completely abolished activation of the cres gene promoter. These data suggest that in gonadotroph cells, both C/EBP sites in the cres promoter are critical for gene activation.



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  		FIG. 7. Transient transfection of LßT2 gonadotroph cells with wild-type and mutant cres-pGL3-Basic vectors. A) Schematic representation of wild-type (135 bp) and mutated (1M, 2M, DM) cres-pGL3-Basic constructs compared with that of empty firefly luciferase vector pGL3-Basic. Solid circle, wild-type C/EBP site; X, mutated C/EBP site. B) LßT2 gonadotroph cells were transfected with 2 µg of wild-type or mutant cres-pGL3-Basic vector and 50 ng pRL-TK Renilla luciferase to determine transfection efficiency. Cells were harvested after 48 h and assayed for luciferase activities using the Dual-Luciferase kit (Promega). Control cells were transfected with empty pGL3-Basic vector and pRL-TK Renilla. Data are reported as the ratio of firefly to Renilla luciferase (L/R) and represent the mean ± SEM of four independent experiments. 135 bp, wild-type cres promoter; 1M, mutated cres C/EBP site 1; 2M, mutated cres C/EBP site 2; DM, double mutant, cres C/EBP sites 1 and 2 mutated

Activation of the Cres Promoter by C/EBPß Protein

To determine whether C/EBPß protein transactivates the cres promoter in a defined cell system, HeLa and HepG2 cells, which do not express endogenous cres (unpublished observations) and little to no C/EBPs [25], were cotransfected with the wild-type and mutated cres-pGL3-Basic vectors and the pMEX-C/EBPß expression vector for introduction of C/EBPß protein. In the presence of increasing amounts of C/EBPß expression vector, a dose-dependent increase in the activation of the wild-type cres promoter (135 bp) was observed in HeLa and HepG2 cells (Fig. 8, A and B, respectively). Western blot analysis of C/EBPß protein in transfected cell lysates, shown below the luciferase data, demonstrate that with increasing amounts of expression plasmid transfected, increasing amounts of C/EBPß protein were detected. Peak activation of the cres gene promoter with the maximal stimulatory dose of C/EBPß resulted in 16-fold (HeLa) and 3-fold (HepG2) higher levels of activation over those observed in the absence of C/EBPß protein. Mutation of the cres C/EBP site 1 (1M) to a nonconsensus C/EBP site lowered the transactivation of the cres promoter by 2-fold to 3-fold in both cell systems, whereas mutation of the cres C/EBP site 2 (2M) resulted in an even more profound decrease in activation of the cres promoter. Finally, mutation of both C/EBP sites (DM), resulted in a loss of activation even in the presence of maximal levels of C/EBPß protein. These studies further demonstrate that C/EBP sites 1 and 2 are important for cres gene activation.



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  		FIG. 8. Transient transfection of HeLa and HepG2 cells with cres-pGL3-Basic and C/EBPß expression vectors. A) HeLa and (B) HepG2 cells were cotransfected with 200 ng wild-type (135 bp) or mutated (1M, 2M, DM) cres-pGL3-Basic vector and 0, 100, 150, and 300 ng of C/EBPß expression vector, bars 1–4, respectively. Cells were incubated for 48 h and then harvested and assayed for firefly luciferase activity. Western blot analysis of the same cell lysates demonstrated increasing amounts of C/EBPß protein in cells transfected with increasing amounts of C/EBPß expression vector (as indicated below the luciferase data). Data are expressed as relative light units (RLU) and represent the mean ± SEM of four independent experiments

DISCUSSION

The cres gene is primarily expressed in the proximal caput epididymidis, stage-specific germ cells in the testis, and the anterior pituitary gonadotroph cells [1, 3, 4]. To begin to identify DNA binding proteins necessary for basal as well as cell- and tissue-specific expression, we initiated studies to examine the cres gene promoter. The identification of two consensus C/EBP sites in the cres promoter as well as in the proximal promoters of other genes expressed in the proximal caput epididymis prompted us to examine the potential significance of C/EBP proteins in the epididymis by studying the interaction of C/EBP family members with the cres promoter. Several lines of evidence suggested that, in particular, C/EBPß protein may be critical for cres gene regulation in both the epididymis and gonadotroph cells, including that C/EBPß is the family member that is predominantly expressed in the gonadotroph cell line LßT2 as well as the epididymis, and that C/EBPß mRNA exhibits region-specific expression in the epididymis and is primarily expressed in the same region as well as the same cell type as the cres gene. Furthermore, our analysis of C/EBPß-deficient mice showed a profound decrease in cres mRNA levels in the mutant epididymis compared with those that are heterozygous, whereas no difference in cres expression was observed between the epididymides of heterozygous and C/EBP{delta}-deficient mice. These observations suggested that, although other yet-to-be-identified transcription factors are also necessary for cres expression, C/EBPß is important for high levels of cres mRNA in the proximal epididymis.

C/EBPß knockout mice are characterized by the presence of immune defects [26, 27] as well as female sterility due to the lack of formation of corpora lutea [10]. Although mating studies indicate that male C/EBPß knockout mice are fertile, detailed analyses of reproductive processes have not been carried out, and thus, whether a subtle or age-related male phenotype exists has yet to be determined. Preliminary histological analyses of the epididymides from young (60- to 120-day-old) animals used in our studies, however, revealed no significant differences between the heterozygous and C/EBPß homozygous null mice, suggesting that the decrease in cres expression observed in the null mouse is not due to a smaller or absent proximal caput region (unpublished observations). In further support of this, the mEP17 gene, which is also expressed in the proximal caput region, showed no difference in mRNA levels between heterozygous and homozygous null mice. Because epididymal cres expression is dependent on testicular factors [1], it is also possible that the C/EBPß null mouse exhibits altered levels of these factors that indirectly could result in decreased cres expression. Although we cannot eliminate the possibility that different proximal caput genes require distinct testicular factors for expression, the observation that mEP17 gene expression, which is also dependent on testicular factors [14], was not different between C/EBPß heterozygous and homozygous null mice, suggests this likely is not the cause of decreased epididymal cres levels. Therefore, our studies suggest a direct effect of C/EBPß on the cres gene promoter. The lack of an effect of the C/EBPß knockout condition on cres mRNA levels in the testis suggests that, in contrast to that in the gonadotrophs and epididymis, C/EBPß may not play a role in cres gene regulation in the testis. Indeed, relative to the epididymis, expression of C/EBPß mRNA in the testis is low and, to date, the C/EBPß gene has been shown to be expressed only in isolated Leydig and Sertoli cells and not testicular germ cells [11, 12]. Therefore, it is likely that transcription factors other than C/EBPß are necessary for cres gene expression in germ cells.

To test whether C/EBPß protein directly bound to the cres gene promoter, EMSAs using recombinant or endogenous C/EBPß protein from nuclear extracts and oligonucleotides representing the two C/EBP sites in the proximal cres promoter were performed. The detection of DNA/protein complexes with both cres C/EBP sites suggested that C/EBPß protein was indeed able to bind to these two consensus sites. Not surprisingly, with both recombinant protein and LßT2 nuclear extracts, C/EBPß protein appeared to bind more strongly to cres C/EBP site 2 than site 1. This is likely due to the presence of a consensus C/EBP half-site in cres C/EBP site 2 compared with a more divergent C/EBP consensus at cres site 1. In contrast to that observed with recombinant protein, several DNA/protein complexes were identified with each C/EBP oligomer when LßT2 nuclear extracts were used as the source of protein, suggesting that C/EBPß may participate in more than one binding species in LßT2 cells.

EMSAs also showed that nuclear proteins present in proximal caput epididymal cells specifically bound to the cres C/EBP sites 1 and 2, whereas supershift assays demonstrated that predominantly C/EBPß and some C/EBP{alpha} protein was present in the DNA/protein complexes. Due to the lack of a positive control for the C/EBP{delta} antibody, however, it is possible that C/EBP{delta} protein may also be present in the DNA/protein complexes. Similar to that observed with LßT2 nuclear extracts, several DNA/protein complexes were detected in the gel shift assays, suggesting that in the epididymis, C/EBPß may also participate in more than one binding species. Interestingly, the binding complexes in the epididymal extracts were different from those in the gonadotrophs. Moreover, in contrast to that observed with the gonadotroph extracts, C/EBPß protein appeared to bind equally if not better to cres C/EBP site 1 than 2. Although these studies indicate that C/EBPß protein is important for cres gene regulation, they also suggest that the presence of unique combinations of transcriptional activators in the gonadotroph and epididymal cells may, in part, contribute to the cell-specific expression of the cres gene.

The functional role of C/EBP sites 1 and 2 in cres gene transactivation was demonstrated by transient transfection assays. Transfection of heterologous cell systems with the cres promoter-luciferase constructs and a C/EBPß expression plasmid showed a dose-dependent increase in transactivation of the cres promoter in the presence of increasing C/EBPß protein and a profound decrease in transactivation when C/EBP sites were mutated, suggesting that C/EBPß protein directly interacts with the cres promoter and enhances transcription. Similarly, transactivation of the cres-luciferase reporter construct in LßT2 cells was effectively inhibited when either cres C/EBP site 1, site 2, or both sites were mutated to nonconsensus C/EBP binding sites. The results of the transfection studies paralleled that of the gel shift studies in that mutation of C/EBP site 1 had less of an effect on cres transactivation than mutation of the C/EBP site 2, which suggests that, similar to that observed in vitro, C/EBPß binding may also be stronger to site 2 than site 1 in vivo.

The studies presented herein demonstrate that the C/EBPß transcription factor plays a necessary role in cres gene activation in the epididymis and gonadotroph cells. To date, little information is known regarding cell-specific expression in the anterior pituitary gonadotroph cells, and even less is known regarding cell-specific expression in the epididymis. Pit-1 is a pituitary-specific transcription factor necessary for expression of genes in somatotrophs, lactotrophs, and possibly thyrotrophs [28, 29], whereas SF-1 is important for conferring gonadotroph-specific expression of several genes [30, 31]. C/EBP family members may also be involved in pituitary-specific regulation of genes as evidenced by studies that showed C/EBP{alpha} was important for the pituitary-specific regulation of the growth hormone gene [16]. Our studies of the cres gene suggest that C/EBPß may also be important for high levels of gene expression in the gonadotroph cells. Several lines of evidence suggest that C/EBPß may synergize with other promoter-bound transcription factors, in particular, members of the steroid hormone receptor superfamily such as the estrogen and glucocorticoid receptors, to activate transcription of specific genes [32, 33]. Indeed, studies of the StAR gene promoter demonstrated that two C/EBP sites were required for SF-1-dependent transactivation of the StAR promoter, whereas studies of the growth hormone promoter suggest that C/EBP{alpha} may synergize with Pit-1[16, 34]. The presence of an SF-1 site as well as several EREs in the cres gene promoter suggests that these factors may also be critical for cres gene expression, possibly through synergistic action with C/EBPß. This has yet to be tested.

Transcription factors that play necessary roles in gene regulation in the epididymis thus far have been limited to studies of the PEA3 transcription factor. PEA3 exhibits highly regionalized expression and is restricted to the proximal caput epididymal region and, similar to other proximal caput epididymal expressed genes, is regulated by unknown factors from the testis [6, 35]. The presence of PEA3 consensus sites in the promoters of several proximal caput expressed genes including GGT, GPX, E-RABP, and CRISP1 suggest that this transcriptional regulatory protein may carry out important roles in the proximal caput epididymal region. The absence of PEA3 sites in the cres gene promoter, however, also suggests that PEA3 regulates only a subset of proximal caput epididymal genes. Also, to date, a direct binding of PEA3 and transactivation of the gene promoter has been demonstrated only for the GGT [35] and GPX genes [35]. Other transcription factors that are expressed in the epididymis include B-myc [24], Pem [37], and Pax-2 [38]. However, no direct interactions of these DNA binding proteins with their cognate promoters have been demonstrated.

The studies presented here show that C/EBPß is a transcription factor that is necessary for high levels of cres gene expression in the proximal caput epididymal region. Although the importance of C/EBP in regulating other proximal caput epididymal expressed genes awaits further study, the regionalized expression of C/EBPß mRNA in the epididymis and the presence of consensus C/EBP sites in promoters of other proximal caput epididymal expressed genes suggests that, like PEA3, C/EBPß may be an important transcriptional regulatory protein of a subset of genes in this epididymal region. Additional studies of the cres gene promoter in transient transfection assays as well as transgenic approaches will ultimately allow us to identify DNA elements and the regulatory proteins that bind, which together, direct cell- and tissue-specific expression of the cres gene.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Dr. Peter Johnson for his contribution of C/EBP knockout mice, and Dr. Simon Williams for his contributions of C/EBP cDNAs and tissues from C/EBP knockout mice as well as his helpful discussions throughout this study. The authors also thank Dr. Curt Pfarr for his helpful suggestions and Dr. Stephen R. Hann for his critical reading of the manuscript.

FOOTNOTES

First decision: 12 June 2001.

1 This work was supported by National Institutes of Health (NIH) grant HD33903 and by a TTUHSC Seed Grant (both to G.A.C). N.H. was supported by NIH grant T32-HD07271. Back

2 Correspondence: Gail A. Cornwall, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street, Lubbock, TX 79430. FAX: 806 743 2990; gail.cornwall{at}ttmc.ttuhsc.edu Back

Accepted: June 26, 2001.

Received: May 10, 2001.

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J Androl, November 1, 2006; 27(6): 802 - 815.
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N. Hsia, J. P. Brousal, S. R. Hann, and G. A. Cornwall
Recapitulation of Germ Cell- and Pituitary-Specific Expression With 1.6 kb of the Cystatin-Related Epididymal Spermatogenic (Cres) Gene Promoter in Transgenic Mice
J Androl, March 1, 2005; 26(2): 249 - 257.
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J. L. Kirby, L. Yang, J. C. Labus, R. J. Lye, N. Hsia, R. Day, G. A. Cornwall, and B. T. Hinton
Characterization of Epididymal Epithelial Cell-Specific Gene Promoters by In Vivo Electroporation
Biol Reprod, August 1, 2004; 71(2): 613 - 619.
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P. Sipila, R. Shariatmadari, I. T. Huhtaniemi, and M. Poutanen
Immortalization of Epididymal Epithelium in Transgenic Mice Expressing Simian Virus 40 T Antigen: Characterization of Cell Lines and Regulation of the Polyoma Enhancer Activator 3
Endocrinology, January 1, 2004; 145(1): 437 - 446.
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T. G. Cooper, A. Wagenfeld, G. A. Cornwall, N. Hsia, S. T. Chu, M.-C. Orgebin-Crist, J. Drevet, P. Vernet, C. Avram, E. Nieschlag, et al.
Gene and Protein Expression in the Epididymis of Infertile c-ros Receptor Tyrosine Kinase-Deficient Mice
Biol Reprod, November 1, 2003; 69(5): 1750 - 1762.
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N. Hsia and G. A. Cornwall
Cres2 and Cres3: New Members of the Cystatin-Related Epididymal Spermatogenic Subgroup of Family 2 Cystatins
Endocrinology, March 1, 2003; 144(3): 909 - 915.
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K. G. Hamil, Q. Liu, P. Sivashanmugam, S. Yenugu, R. Soundararajan, G. Grossman, R. T. Richardson, Y.-L. Zhang, M. G. O'Rand, P. Petrusz, et al.
Cystatin 11: A Ne