| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
c Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430
d Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2175
ABSTRACT
The myc family of transcriptional regulators carries out critical roles in the control of cellular proliferation, differentiation, apoptosis, and tumorigenesis. The B-myc gene is a recently identified myc family member that has not been well characterized. Previously, we have shown that B-Myc inhibits the ability of c-Myc to transform cells and can inhibit cellular proliferation. Because B-myc is primarily expressed in hormonally regulated tissues with predominant expression in the epididymis, we examined in greater detail B-myc expression in the epididymis to ultimately understand potential roles B-myc may play in this and other hormonally regulated tissues. Herein we demonstrate that, in contrast to c-myc, B-myc mRNA and protein expression are highly regionalized with expression predominantly in the proximal caput epididymal region. Furthermore, in situ and immunohistochemical analyses show that within the epididymis B-myc mRNA and protein are specifically expressed by the epithelial cells and that B-Myc protein is localized to both the nuclear and cytosolic compartments. Castration and hormone replacement studies further show that expression of the B-myc mRNA is highly dependent on the presence of androgens and testicular factors. Finally, mRNA turnover studies demonstrate that the B-myc mRNA is relatively unstable with a half-life of 3.5 h. Taken together, the highly restricted and regulated expression of the B-myc gene suggests it may play important regulatory roles in the epididymis and perhaps other hormonally regulated tissues.
epididymis, gene regulation, sperm maturation, testosterone
INTRODUCTION
The myc family of transcriptional regulators, including c-myc, N-myc, L-myc, S-myc, and B-myc, is thought to play critical roles in the control of cell proliferation, differentiation, apoptosis, and tumorigenesis [1, 2]. The most well-studied family member is c-myc, which is a powerful stimulator of cell proliferation and likely exerts its effects via the regulation of genes involved in cell cycle progression. Indeed, overexpression of c-myc not only stimulates cell proliferation but also inhibits differentiation of several cell types and can lead to transformation and tumorigenesis [1, 2]. The c-myc gene is characterized as a member of the helix-loop-helix leucine zipper (bHLH/LZ) class of transcription factors and contains an N-terminal transactivation domain as well as a C-terminal bHLH/LZ DNA binding domain, both of which are critical for c-Myc function [1]. c-Myc acts as a transcriptional regulator by binding to specific DNA sequences with its partner Max [3]. Following specific interactions with factors binding to its N-terminal transactivation domain, the c-Myc/Max complex is then thought to regulate specific genes involved in cell proliferation [3, 4].
The B-myc gene is a more recently described myc family member that, to date, has not been well characterized. The B-myc gene is structurally and functionally distinct from the other myc genes and contains only a single coding exon, which is similar to c-myc exon 2. The B-Myc protein does not contain a bHLH/LZ DNA binding domain or nuclear localization signals but does contain a transactivation domain [5]. Furthermore, unlike other myc genes, B-Myc has been shown to inhibit c-Myc function in the Myc/Ras cotransformation assay [6]. The c-Myc-inhibiting activity of B-Myc suggests it may be a potentially important myc family member. However, because previous reports showed low levels of B-myc mRNA in several tissues, with highest levels in brain [5] and a lack of regulation of expression by growth factors and during differentiation of F9 cells [7], further studies on B-myc expression and function had not been pursued. Indeed, the biological role of B-Myc has not yet been determined. Herein, we show that the B-myc gene is abundantly expressed in the mouse epididymis with less expression in the prostate gland and brain. These observations, taken together with our other studies showing B;nhmyc mRNA and protein expression in adrenal gland, pituitary gland, mammary gland, ovary, and uterus [8] suggest that B-myc gene expression is associated with hormonally regulated tissues. Our studies also showed that B-Myc inhibits cellular proliferation [8]. Therefore, B-Myc may be an important modulator of cell proliferation in hormonally regulated tissues. The focus of the present studies was to examine in greater detail B-myc expression and its regulation by hormones in the epididymis to ultimately gain insight toward putative roles this gene may play in the epididymis and other hormonally regulated tissues.
MATERIALS AND METHODS
Experimental Animals
Mature male and female ICR mice were purchased from Charles River (Wilmington, MA). All mice were housed under a constant 12L:12D cycle and were allowed free access to food and water. Orchiectomies and efferent duct ligations were carried out by the abdominal route after injection of ketamine/xylazine. Testosterone (T) replacement was by the implantation of a 5-mg T pellet (Innovative Research Of America, Toledo, OH) directly under the skin on the back of each mouse. Dihydrotestosterone (DHT) (Sigma, St. Louis, MO) in sesame oil was administered by daily s.c. injections of 25 µg/day/mouse. At the time mice were killed the relative size of the seminal vesicles was noted as an indicator of the presence or absence of circulating androgens. Furthermore, the remainder of the T pellet was isolated from each implanted mouse to ensure that it had not been lost during the replacement or maintenance period. Blood was collected at the time of killing for RIA determination of circulating T and DHT levels in the hormonally manipulated mice. After clotting, the serum was collected and stored at -20°C until assayed. RIAs were performed by the Center for Reproductive Biology Research Endocrine core facility, Vanderbilt University. All animal studies were conducted in accord with the principles and procedures outlined in the NIH Guidelines for Care and Use of Experimental Animals.
Cell Culture
The gonadotroph cell line LßT2 was a generous gift of P. Mellon, University of California, San Diego, CA. LßT2 cells were cultured in 100-mm tissue culture dishes and were maintained in Dulbecco modified Eagle medium (DMEM) with 4.5 mg/ml glucose, 5% fetal calf serum, 5% calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37°C in an atmosphere of 5% CO2. Media and antibiotics were from Gibco BRL (Grand Island, NY) and serum was from Hyclone (Logan, UT).
Isolation of RNA
Total RNA was isolated from mouse tissue using Trizol reagent (Gibco BRL) following the manufacturer's protocol. For the isolation of RNA from cultured cells, medium was aspirated from tissue culture plates, the plates were rinsed quickly with cold sterile PBS, and 5 ml Trizol was added directly to each plate. The cells were resuspended by scraping the plate with a sterile spatula followed by repeated pipetting and isolation of RNA.
Northern Blot Analysis
Total RNA from mouse tissues and cell lines was separated on a 1% agarose gel containing borate buffer, pH 8.2, and 0.66 M formaldehyde. The RNA samples were heated at 95°C for 2 min and then loaded onto the gel and electrophoresed. To verify equal loading of RNA in each lane of the gel, ethidium bromide was included in the RNA sample. The gels were washed extensively in water to remove formaldehyde before transferring to nylon membrane (Nytran; Schleicher and Schuell, Keene, NH). The blots were prehybridized for 2 h at 42°C in hybridization buffer containing 50% formamide, 5x SSC (0.075 M sodium citrate, 0.75 M NaCl, pH 7.0), 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 105 cpm probe/ml hybridization buffer. Blots were sequentially probed with 32P-labeled B-myc, c-myc [8], and 18S cDNAs prepared using a random primer labeling method (Prime-It; Stratagene, La Jolla, CA). After hybridization, the blots were washed in 2x SSC (0.03 M sodium citrate, 0.3 M NaCl, pH 7.0) at room temperature for 10 min followed by washing in 2x SSC, 1% SDS at 42°C for three times 15 min each and then 1–2 times for 15 min at 65°C before exposure to film. Northern blots were repeated two to four times using mRNA from two to three different preparations, and representative blots are presented.
To quantititate the total amount of RNA in each lane of the Northern blots, the autoradiograms were scanned using a computer-assisted image analysis system (BioImage Visage 2000; BioImage, Ann Arbor, MI). The integrated areas obtained for the B-myc, c-myc, and Cres (cystatin-related epididymal spermatogenic) probes were normalized to that for the 18S ribosomal probe.
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, Pittsburgh, PA) 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% polyvinylpyrrolidone, 0.02% BSA), 10% dextran sulfate, and 10 mM dithiothreitol (DTT), and approximately 4 x 106 cpm of an 35S-labeled antisense B-myc RNA probe. To determine the specificity of the labeling, epididymal sections were hybridized under the conditions described above but with the sense-strand B-myc RNA probe. 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 (1.5 mM sodium citrate, 15 mM NaCl, pH 7.0), 10 mM ß-mercaptoethanol, 1 mM EDTA at 55°C with three changes after 5, 20, 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%, 100%) containing 0.3 M NH4Ac. The slides were dipped in Kodak NTB-2 emulsion, dried, and exposed for 10–14 days. The exposed slides were developed, fixed, and stained with hematoxylin and eosin, and coverslips were applied with Permount.
Messenger RNA Stability Studies
LßT2 gonadotroph cells were cultured in DMEM at a density of 3 x 106 cells/100-mm plate. To inhibit RNA synthesis, actinomycin D (Sigma) in 100% ethanol was added to a final concentration of 10 µg/ml, and cells were harvested at Time 0 (immediately after addition of actinomycin D), 1, 4, 10, and 24 h. Control cells received an equal volume of ethanol only and were harvested at the same time points. The RNA was isolated from cultured cells as described above using Trizol reagent following the manufacturer's protocol. 3H-Uridine was added (100 µCi/plate) to cultured LßT2 cells incubated in the presence or absence of actinomycin D, and precipitable counts were determined to confirm that RNA synthesis was inhibited by the presence of actinomycin D. These studies showed that RNA synthesis after 2, 8, and 20 h actinomycin D treatment was 23%, 17%, and 3% of control, respectively.
Western Blot Analysis
Mouse tissues were lysed by polytron in radioimmunoprecipitation assay buffer containing 10 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.2% SDS, 0.1 mg/ml PMSF, and 0.01 mg/ml aprotinin. One hundred micrograms of total protein was separated on a 10% SDS polyacrylamide gel and transferred to nitrocellulose. The blot was incubated in Tris-buffered saline (TBS) (50 mM Tris-Cl, pH 7.4, 200 mM NaCl) containing 3% milk to block followed by 10 µg/ml affinity-purified B-Myc antibody in TBS, 3% milk overnight at 4°C. Blots were then washed in TBS at room temperature followed by incubation in TBS, 3% milk containing horseradish peroxidase-conjugated donkey antirabbit secondary antibody (1:30 000) (Jackson ImmunoResearch Laboratories, West Grove, PA). Blots were washed extensively in TBS, incubated with chemiluminescent substrate (ECL; Amersham Pharmacia Biotech Inc., Piscataway, NJ), and exposed to film.
Immunohistochemistry
Paraformaldehyde-fixed, paraffin-embedded epididymal tissue sections were deparaffinized and rehydrated by incubation in xylene followed by 100%, 95%, 70%, 50% ethanol. Antigen retrieval was carried out by microwaving slides on high in 0.01 M sodium citrate buffer, pH 6, for two times 5 min each. Sections were then washed in PBS containing 0.1% Triton X-100 (PBST), and endogenous peroxidase activity was inhibited by incubating slides in 100% methanol with 0.3% hydrogen peroxide for 20 min. Slides were washed two times 10 min each in PBST followed by incubation for 1 h in a humidified box in PBST containing 2.5% normal goat serum to block. The B-Myc antiserum diluted 1:1000 in PBST containing 1% normal goat serum was added to each section and incubated overnight at 4°C. The following day, sections were washed three times 10 min each in PBST and incubated for 1 h with a biotinylated secondary antibody (Pierce Chemical Co.) diluted 1:200 in PBST with 1% normal goat serum. Sections were then incubated with the ABC reagent (Pierce Chemical Co., Rockford, IL) for 2 h, washed in PBST, and incubated with 0.2 mg/ml 3,3'-diaminobenzidine (DAB) in 0.1 M Tris-Cl, pH 7.2, containing 0.01% hydrogen peroxide until the reaction was complete. Slides were washed in water to stop the reaction, counterstained with hematoxylin/eosin, dehydrated, and mounted with Permount.
RESULTS
Tissue-Specific Expression of B-myc mRNA
Previous studies showed that the B-myc gene was expressed at low levels in several rat tissues including lung, thymus, kidney, heart, liver, and spleen with highest levels of expression in the brain [5]. Because these investigators limited their analyses to somatic tissues, we examined B-myc expression in several male reproductive tissues by Northern blot analysis. As shown in Figure 1, the 1.3-kb B-myc mRNA was predominantly expressed in the mouse epididymis with lower levels of expression in brain, prostate gland, and thymus. The B-myc mRNA was very low or undetectable in vas deferens, testis, liver, and kidney. In contrast to B-myc, the 2.2-kb c-myc mRNA was primarily expressed in thymus with lower levels of expression in the prostate gland, epididymis, and vas deferens, and little or no mRNA in brain, testis, liver, and kidney. These results show that within these tissues, B-myc and c-myc expression are distinct. Furthermore, these observations, taken together with other studies showing B-myc expression in the ovary, uterus, mammary gland, adrenal gland, and pituitary gland [8], suggest that B-myc expression is primarily associated with hormonally regulated tissues.
|
Regionalized Expression of B-myc mRNA in the Mouse Epididymis
Many genes in the epididymis exhibit highly regionalized expression that is thought to be critical for epididymal function [9]. To determine if the B-myc gene is expressed in a regional-specific manner, Northern blot analysis was performed using total RNA isolated from five epididymal regions. Expression of c-myc in the different epididymal regions was also examined to determine if a correlation exists between these two family members. As shown in Figure 2, B-myc mRNA was highly regionalized and was predominantly expressed in the proximal caput epididymidis (region 1) with decreasing levels of expression in the distal epididymal regions. In contrast, low levels of the c-myc mRNA were detected in all epididymal regions. In addition, little B-myc and no c-myc mRNA were detected in the mouse testis (Figs. 1 and 2).
|
In situ hybridization was next performed to identify the specific epididymal cell population expressing the B-myc gene. Figure 3 shows bright-field and dark-field exposures of a longitudinal section of the mouse epididymis hybridized with antisense and sense B-myc RNA probes. Silver grains representing B-myc mRNA were distributed throughout the epididymal epithelium with highest levels of B-myc expression detected in the tall columnar epithelium of the proximal caput epididymidis (Fig. 3, A and B) and lower levels of expression in the cuboidal epithelium of the cauda epididymidis (Fig. 3, C and D). Hybridization of epididymal tissue with the control, sense B-myc RNA probe showed only a low background signal that was similar between the proximal caput (Fig. 3, E and F) and cauda epididymal regions (not shown). Although the in situ studies show B-myc mRNA to be localized to the epididymal epithelial cells, known as the principal cells, we cannot rule out that other minor cell types may also express the B-myc gene.
|
Regionalized Expression of B-Myc Protein in the Mouse Epididymis
We have shown that B-Myc protein is present in mouse tissues as a 26-kDa protein [8]. Western blot analyses were performed using an affinity-purified B-Myc antibody to determine if expression of B-Myc protein in the epididymis is region specific. As shown in Figure 4A, high levels of B-Myc protein were present in the epididymis compared to the levels in brain and vas deferens. Similar to the B-myc mRNA, the B-Myc protein was also regionally expressed and was predominantly found in the proximal caput epididymal region (region 1) with decreasing levels of protein present in the distal epididymal regions. The specificity of the antibody for the B-Myc protein was shown by blocking experiments in which the antibody was preincubated with recombinant B-Myc protein prior to using in the Western blots. No specific signal was detected in these Western blots (Fig. 4B).
|
To examine further B-Myc protein localization in the epididymis immunohistochemical analyses were performed. Epididymal tissue sections were incubated with mouse B-Myc antiserum followed by a biotinylated secondary antibody and DAB staining. These studies showed that B-Myc protein was primarily found in the epithelium of the proximal caput epididymal region with decreasing levels of protein detected in the distal epididymal regions (Fig. 5A), thus confirming the in situ and Western blot studies. Furthermore, within the epididymal epithelial cells immunoreactivity was detected in the basally localized nuclei as well as the cytosol indicating that B-Myc protein is localized to both subcellular compartments (Fig. 5, B and C). Epididymal tissue sections incubated with preimmune serum showed only a low background level of immunoreactivity (Fig. 5D). These studies confirm our subcellular fractionation analysis of B-Myc in COS-7 cells [8].
|
Androgen Regulation of B-Myc Gene Expression
Because epididymal morphology and function are highly androgen dependent, Northern blot analysis was carried out to determine if B-myc expression in the epididymis is dependent on the presence of androgens. Total RNA was isolated from epididymides pooled from mice that were intact or bilaterally castrated for varying lengths of time. Compared to intact (I) animals, within 12 h of T withdrawal by castration, B-myc mRNA levels in the epididymis had dramatically decreased by 67% and continued to decrease over the castration time course to levels approximately 5% of intact levels after 1 wk castration (Fig. 6). In striking contrast to the decrease in B-myc expression following castration, c-myc expression in the epididymis showed an immediate threefold increase that recovered to intact levels within 3 days (Fig. 6). The dramatic decrease in B-myc mRNA levels following castration suggests that B-myc expression in the epididymis is highly regulated by the testis.
|
Experiments were next carried out to determine if the administration of exogenous androgens to castrated mice restored B-myc expression in the epididymis. The administration of T (T-rpl) or its 5
-reduced metabolite DHT, the active androgen in the epididymis, to mice that had been castrated for 1 wk resulted in a partial recovery of B-myc mRNA to levels 30%–40% of that in the intact animal (Fig. 6). To eliminate the possibility that 1 wk castration caused the down-regulation of androgen receptors or other necessary proteins in the epididymis that could not be induced by T and thus could prevent the maximal effects of exogenous androgen on B-myc expression, T was administered to mice at the time of castration in a T maintenance paradigm. However, B-myc mRNA levels in the epididymides of T-maintained (T-mn) mice were reduced and were only 35% of that in the intact animals (Fig. 6). The RIA analysis showed that circulating levels of T and DHT were approximately 16-fold and 6-fold higher, respectively, than control intact mice (Fig. 6 legend), suggesting that the incomplete recovery of B-myc expression in the epididymis following castration and hormone replacement was not due to insufficient androgens. In contrast to B-myc, c-myc expression in the epididymis was unaffected by the presence of exogenous androgens (Fig. 6). Taken together, these studies suggest that the down-regulation of B-myc is due in part to the withdrawal of androgens because T or DHT administration resulted in a partial recovery of B-myc mRNA levels. However, these studies also suggest that, in addition to androgens, other factors from the testis are necessary for the maximal expression of the B-myc gene in the epididymis.
Testis Regulation of B-Myc Gene Expression
To date, several genes in the proximal caput epididymidis have been shown to require the presence of unknown factors from the testis for expression [9]. To address this possibility, several experimental approaches were used to determine the effect of testicular factors on B-myc expression in the epididymis. First, to prevent testicular fluid from entering the epididymis without disrupting blood flow and thus the presence of circulating androgens, the efferent ducts that connect the testis and epididymis were ligated for 1 wk. As shown in Figure 7, the epididymides of mice that underwent efferent duct ligation (EDL) and therefore did not receive input from the testis showed an 85% reduction in the level of B-myc expression compared to that in nonligated, intact (I) epididymides. Also to examine the effects of testicular factors on B-myc expression in the epididymis, mice were unilaterally castrated (UC) for 1 wk. In this experimental condition, the loss of one testis causes the contralateral testis to increase its secretion of T and therefore, as shown by RIA (Fig. 7 legend), the circulating levels of T in these mice were comparable to that of intact mice. Similar to efferent duct ligated mice, examination of UC mice allows us to assess the effect of the loss of testicular factors on B-myc expression in the epididymis while maintaining normal levels of circulating androgens. As shown in Figure 7, the epididymis that lacked the presence of a testis (UC mice-castrate epididymis) (UC-C) exhibited 85% lower levels of B-myc mRNA compared to B-myc mRNA levels in the epididymis that remained in contact with the testis (UC mice-intact epididymis) (UC-I). In contrast to the observed changes in B-myc expression, both efferent duct ligation and unilateral castration experiments did not alter c-myc expression in the epididymis.
|
In a second approach to examine the effects of testicular factors on B-myc expression in the epididymis, mice that have impaired testicular function due to a specific genetic mutation were examined. Mice that are homozygous for the c-kit gene mutation (white spotting locus mutation) are germ cell deficient. The c-kit mutant mice, however, appear to have normal Sertoli, Leydig, and peritubular cells [10, 11], and circulating and tissue T levels in these mice are not different from wild-type mice [12]. As shown in Figure 7, mice that were homozygous germ cell deficient (-/-) showed an 80% reduction in the levels of B-myc mRNA in the epididymis compared to that in homozygous wild-type (+/+) and heterozygous (±) mice. In contrast, compared to wild-type mice, c-myc mRNA levels increased by approximately threefold in the heterozygous and homozygous c-kit mutant mice.
Stability of the B-myc mRNA in LßT2 Gonadotroph Cells
Because there are no epididymal cell lines available, the LßT2 gonadotroph cell line that expresses both the B-myc and c-myc mRNAs [8] was selected to determine the relative half-life of the B-myc mRNA compared to c-myc mRNA. Cultured LßT2 cells were incubated in the presence and absence of the RNA synthesis inhibitor actinomycin D and cells were harvested over a 24-h period. The c-myc mRNA showed a greater than 50% decrease in total mRNA levels after 1-h actinomycin D treatment (Fig. 8). After 4- and 10-h exposure to actinomycin D, B-myc levels had decreased by 63% and 93%, respectively. These results predict the half-life of B-myc mRNA to be 3.5 h approximately. The 3H-uridine uptake experiments confirmed that RNA synthesis was inhibited by the presence of actinomycin D (see Materials and Methods). To eliminate the possibility that reduced B-myc mRNA levels in LßT2 cells after 4- and 10-h actinomycin D treatment reflected cell death, the stability of another gene was also examined. As shown in Figure 8, steady-state levels of the Cres mRNA [13] were unaffected after 10 h actinomycin D treatment.
|
DISCUSSION
Regional and Cell-Specific Expression of B-myc mRNA
We have demonstrated that the B-myc mRNA and protein are abundantly expressed in hormonally regulated tissues with the highest levels of expression in the mouse epididymis. In addition to exhibiting tissue-specific expression, the studies presented here show that within the epididymis, B-myc mRNA and protein are highly restricted to the proximal caput epididymal epithelium with decreasing levels of expression in the distal epididymal regions. These observations suggest that B-Myc function may not only be tissue specific but may be region and cell specific as well. Because these studies are the first to examine in vivo the cell type that expresses the B-myc gene and protein, we cannot rule out that B-myc may also be expressed by different cell types in other tissues. However, preliminary examination of B-myc expression in mouse and rat prostate tissue also showed mRNA in the epithelial cells (unpublished observations).
Within the epididymal epithelial cells, intense immunoreactivity was detected in the nucleus with less immunoreactivity in the cytosol suggesting that B-Myc protein is localized to both subcellular compartments. These observations are consistent with our subcellular fractionation analysis of COS-7 cells overexpressing B-Myc [8] as well as those of Asker et al. [14] who examined the intracellular localization of overexpressed B-Myc in transfected DG75 Burkitt lymphoma cells. Our immunohistochemical studies suggest, therefore, that the nuclear localization of B-Myc is not an artifact of overexpression in cell lines. Because B-Myc lacks nuclear localization signals, it is likely that the binding of B-Myc to other nuclear-targeted proteins is necessary for its translocation to the nucleus. Furthermore, translocation to the nucleus by binding to other nuclear proteins could be a means of regulating B-myc function.
Regulation of B-myc Gene Expression
Initial studies of B-myc gene regulation in murine embryonal carcinoma cells suggested that, in contrast to other myc family members, B-myc expression was unaffected by serum starvation or growth factor stimulation or during the induction of differentiation by retinoic acid [7]. Recently, however, studies in the developing mouse embryo showed that the B-myc gene was strongly induced in the trophectoderm lineage as the cells began to differentiate, suggesting that B-Myc may negatively regulate cellular proliferation and may be associated with differentiation [15]. Also, studies of c-myc expression in the rainbow trout identified a putative B-myc mRNA in the pituitary gland that was upregulated during sexual maturation [16].
Our studies demonstrate that in the epididymis B-myc expression is highly regulated by steroid hormones and testis factors and may be associated with a differentiated cell phenotype. The epididymis is a nonproliferating, highly differentiated organ that is critically dependent on androgens for maintaining normal cellular morphology and function. Following the withdrawal of androgens, the epididymal epithelium involutes, genes are down-regulated, and normal sperm maturation does not occur. The administration of androgens reverses, for the most part, the castrate state, and the epididymal epithelium regains its normal morphology and expression of genes. Androgen withdrawal resulted in a dramatic decrease in B-myc expression in the epididymis that recovered, in part, following the administration of T or DHT. While the decrease in B-myc mRNA following castration suggests that the B-myc gene is regulated by steroid hormones, the incomplete recovery of B-myc mRNA levels following hormone replacement suggests that full expression of B-Myc in the epididymis also requires the presence of other testicular factors. This hypothesis is supported by additional studies including efferent duct ligation and unilateral castration that showed that B-myc expression was reduced under conditions in which circulating T levels were normal, but the epididymis was no longer in contact with the testis. Further support for the regulation of B-myc expression by testis factors was provided by the c-kit mutant mouse studies showing a decrease in B-myc expression in these mice with impaired testicular function. The regulation of the proximal caput epididymal region by factors from the testis in addition to androgens has been postulated for some time based on early histological observations and more recently by gene expression studies. Following the administration of androgen to a castrate epididymis, the epithelium recovers to its precastrate morphology in all regions except for the proximal caput/initial segment region [17]. Furthermore, in addition to B-myc, several other genes that are predominantly expressed in the proximal caput epididymidis including Cres [13], Adam7 [18], proenkephalin [19], and gamma glutamyl transpeptidase [20], have been shown to require unknown testicular factors exclusively or in combination with androgens for full expression.
The loss of B-myc expression in the castrate epididymis, conditions under which the epididymis loses its highly differentiated morphology and function, implies that B-myc function is associated with a differentiated cell function. Interestingly, while c-myc expression in the epididymis transiently increased following hormone withdrawal, other experimental conditions that altered B-myc mRNA levels did not affect c-myc expression. These observations suggest that the mechanisms by which c-myc and B-myc gene expression are regulated in the epididymis are distinct. The transient increase in c-myc expression following androgen withdrawal suggests that, in contrast to B-myc, c-myc expression in the epididymis is repressed by androgens. These observations are consistent with other reports that showed in prostate cell lines a transcriptional repression of c-myc in the presence of androgens [21] and an elevation of c-myc mRNA in carcinomas grown in castrated rats [22]. The c-myc also appears to be distinct from B-myc in its upregulation of mRNA levels in mice that are heterozygous and homozygous for the c-kit gene mutation. However, c-myc expression in the epididymis was unaffected in the EDL and UC mice, suggesting that the loss of c-kit rather than the general loss of testicular factors affects c-myc expression in the epididymis.
Stability of B-myc mRNA
Actinomycin D inhibition of RNA synthesis in the LßT2 gonadotroph cells showed that B-myc mRNA was relatively unstable and exhibited a half-life of approximately 3.5 h. Previously the stability of the B-myc mRNA was examined in proliferating F9 embryonal carcinoma cells and was shown to be approximately 6 h [7]. The difference between our observations and those previously reported may reflect the cell system used. The validity of the turnover experiments was confirmed by our analysis of other mRNAs, including those for the c-myc and Cres genes. Our studies show the half-life of the c-myc mRNA to be approximately 30 min, which is in good agreement with previous reports predicting its half-life to be 20–40 min [23]. The relatively unstable nature of B-myc mRNA coupled with the rapid turnover of B-Myc protein [8] is necessary for the rapid modulation of B-myc levels by hormones and testicular factors. This relatively rapid turnover is often observed in other transcriptional regulatory genes.
Implications for B-myc Function in the Epididymis
The c-myc gene has been implicated in a variety of cellular processes including cell proliferation, differentiation, apoptosis, and tumorigenesis. Based on a previous observation that B-Myc can inhibit c-Myc activity in the Myc/Ras cotransformation assay [6], B-Myc has been proposed to function as a regulator of c-Myc. Because B-Myc is structurally similar to the N-terminal transactivation domain of c-Myc, B-Myc may competitively bind limiting factors needed for c-Myc-activated transcription and hence modulate c-Myc function. This hypothesis, while unproven, certainly could be true in tissues where B-myc and c-myc are expressed concurrently. However, in tissues where the two genes are not similarly expressed or regulated, an alternate possibility is that B-Myc may carry out a function that is distinct from that of c-Myc regulation. Indeed, based on the studies presented herein, in the epididymis B-myc may have a function other than modulating c-myc activity. One possibility is that B-Myc may interact with cofactors that are required by transcriptional regulators besides c-Myc that play integral roles in modulating the differentiated functions of the proximal epididymis. Further studies focused on identifying B-Myc-interacting proteins as well as the generation of a B-myc null mouse will provide insight toward understanding the role of B-Myc in the epididymis.
FOOTNOTES
First decision: 9 October 2000.
1 Supported by NIH grants HD33903 (G.A.C.), CA47399 (S.R.H.), and CA78888 (S.R.H.), and the South Plains Foundation (G.A.C.). 
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
Accepted: January 9, 2001.
Received: August 30, 2000.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  E. Prabagaran, U. C. Hegde, S. B. Moodbidri, A. H. Bandivdekar, and V. P. Raghavan Postnatal Expression and Androgen Regulation of HOXBES2 Homeoprotein in Rat Epididymis J Androl, September 1, 2007; 28(5): 755 - 771. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Tomsig and T. T. Turner Growth Factors and the Epididymis J Androl, May 1, 2006; 27(3): 348 - 357. [Full Text] [PDF]
|
|
|
|
|
|
F. Saez, C. Legare, J. Laflamme, and R. Sullivan Vasectomy-Dependent Dysregulation of a Local Renin-Angiotensin System in the Epididymis of the Cynomolgus Monkey (Macaca fascicularis) J Androl, September 1, 2004; 25(5): 784 - 796. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Ruz, S. Andonian, and L. Hermo Immunolocalization and Regulation of Cystic Fibrosis Transmembrane Conductance Regulator in the Adult Rat Epididymis J Androl, March 1, 2004; 25(2): 265 - 273. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Hsia and G. A. Cornwall DNA Microarray Analysis of Region-Specific Gene Expression in the Mouse Epididymis Biol Reprod, February 1, 2004; 70(2): 448 - 457. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Rao, J. C. Herr, P. P. Reddi, M. J. Wolkowicz, L. A. Bush, N. E. Sherman, M. Black, and C. J. Flickinger Cloning and Characterization of a Novel Sperm-Associated Isoantigen (E-3) with Defensin- and Lectin-Like Motifs Expressed in Rat Epididymis Biol Reprod, January 1, 2003; 68(1): 290 - 301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Wassler, P. Syntin, H.G. Sutton-Walsh, N. Hsia, D. M. Hardy, and G. A. Cornwall Identification and Characterization of Cystatin-Related Epididymal Spermatogenic Protein in Human Spermatozoa: Localization in the Equatorial Segment Biol Reprod, September 1, 2002; 67(3): 795 - 803. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. H. Tong and W.-C. Song Estrogen Sulfotransferase: Discrete and Androgen-Dependent Expression in the Male Reproductive Tract and Demonstration of an in Vivo Function in the Mouse Epididymis Endocrinology, August 1, 2002; 143(8): 3144 - 3151. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Hsia and G. A. Cornwall CCAAT/Enhancer Binding Protein {beta} Regulates Expression of the Cystatin-Related Epididymal Spermatogenic (Cres) Gene Biol Reprod, November 1, 2001; 65(5): 1452 - 1461. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
