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From the Departments of Pharmacology & Therapeutics and Obstetrics & Gynecology, McGill University, Montréal, Québec, Canada.
| Correspondence to: Dr Bernard Robaire, McIntyre Medical Sciences Building, 3655 Promenade Sir-William-Osler, Room 104, Montréal, Québec, Canada H3G 1Y6 (e-mail: bernard.robaire{at}mcgill.ca). |
| Received for publication May 24, 2005; accepted for publication August 2, 2005. |
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Abstract |
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Key words: Transcription factor, differential expression, orchidectomy
Inhibitor of DNA binding (Id) genes encode a family of 4 HLH proteins (Id1, Id2, Id3, and Id4) that lack the basic DNA binding domain. Their name is an indicator of their functional properties both as inhibitors of DNA binding and inhibitors of cell differentiation. The family of Id HLH proteins have traditionally been viewed as dominant negative regulators of bHLH transcriptional regulators that lead to cell differentiation by forming transcriptionally inactive, stable dimers with these, thus sequestering them in the cytoplasm (Benezra et al, 1990; Ellis et al, 1990; Garrell and Modolell, 1990). Although Id proteins preferentially bind to class B bHLH proteins, studies have revealed that they may directly interact with and modulate the activities of several non-bHLH transcriptional regulators, such as pRB, ETS-domain transcription factors, and MIDA-1 (Iavarone et al, 1994; Shoji et al, 1995; Yates et al, 1999). Id1, Id2, and Id3 are expressed ubiquitously, whereas Id4 is expressed predominantly in testis, brain, and kidney (Riechmann et al, 1994). Id proteins are essential for cell cycle progression in some cell lines, and they act as positive regulators of cell proliferation (Christy et al, 1991; Norton et al, 1998). In general, Id gene expression is highest in proliferating cells, such as in hematopoietic cell lineages, and lowest in terminally differentiated cells. The role of Id proteins in a terminally differentiated organ such as the epididymis is unknown.
The epididymis is a highly specialized male reproductive organ. It is a narrow, highly convoluted tubule that connects the efferent ducts to the vas deferens and that functions in the transport, maturation, and storage of spermatozoa (Orgebin-Crist, 1967; Robaire and Hinton, 2002). The epididymis is divided morphologically into 4 regions: the initial segment and the caput, corpus, and cauda epididymidis. The epididymal epithelium comprises 5 major epithelial cell types: principal, narrow, clear, basal, and halo cells (Reid and Cleland, 1957; Robaire and Hermo, 1988; Hermo and Robaire, 2002). Principal cells play a major role in secretion and absorption of components of the luminal fluid (Robaire and Hermo, 1988; Hermo et al, 1994). Narrow cells are only found in the initial segment; in the rest of the epididymis these cells give rise to the clear cell, which participates in the uptake of luminal components (Hermo et al, 1988; Vierula et al, 1995; Serre and Robaire, 1999). Basal cells regulate electrolyte and water transport by the principal cells (Leung et al, 2004), while halo cells are thought to be the main type of immune cell in the epididymis (Dym and Romrell, 1975; Flickinger et al, 1997). Therefore, these cell types not only provide structural support but are also integral to the formation of the epididymal fluid, in which sperm acquire motility and fertilizing ability by the time they reach the cauda epididymidis.
We are interested in Id proteins because of the need to understand the complex gene expression along the epididymis. Many genes have been shown to be expressed in a region-specific manner in the epididymis (Cornwall and Hann, 1995; Kirchhoff, 1999; Cornwall et al, 2002). This is thought to account for the regional differences along the epididymis that are essential in the establishment of the environment required for sperm maturation. Many of these genes have been found to decrease in expression from proximal to distal parts of the epididymis (Christy et al, 1991; Jervis and Robaire, 2001). However, studies done in our laboratory using gene expression profiling show that Ids have a particularly noteworthy pattern of expression; Id1, Id2, and Id3 belong to the small group of genes whose expression increases from proximal to distal parts of the epididymis, with the highest expression in the cauda epididymidis (Jervis and Robaire, 2001). This pattern of expression, along with the mechanism by which Id proteins sequester various transcription factors, may therefore provide some insight on the differential expression of other genes along the epididymis that lead to the region-specific differences required for germ cell maturation.
In the present study, we establish the mRNA expression profiles of Id1, Id2, Id3, and Id4 in the initial segment and the caput, corpus, and cauda rat epididymidis. We also determine the immunocytolocalization of the 4 Ids along the epididymis and compare the Id3 protein expression profiles in control, orchidectomized, and aged animals.
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Materials and Methods |
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Bilateral orchidectomy was done through the abdominal route. Efferent ducts were ligated on both sides, and testes were removed above the ligation. Animals were sacrificed at 9 or 35 days postorchidectomy, and tissues were prepared for light microscope (LM) immunohistochemistry. Animals in the control group were sham operated and sacrificed at the same time points postorchidectomy. All animal studies were conducted in accordance with the principles and procedures outlined in the Guide to the Care and Use of Experimental Animals prepared by the Canadian Council on Animal Care (McGill Animal Care Committee protocol 206).
Tissue Preparation for LM Immunohistochemistry
Brown Norway rats and male Sprague-Dawley rats (n = 4) were anesthetized
with an intraperitoneal (IP) injection of anesthetic cocktail of Vetalar
(ketamine HCL, 115.4 mg/mL; Vetrepharm, London, Canada), Anased (xylazine HCL,
20 mg/mL; Novopharm, Toronto, Canada), and Atravet (acepromazine maleate, 10
mg/mL; Ayerst, Montréal, Canada) at a 20:10:1 ratio. The epididymides
were fixed with Bouin solution via perfusion through the abdominal aorta.
Retrograde perfusions were done to obtain optimal fixation of the initial
segment and caput epididymidis, and prograde perfusions were used to fix the
corpus and cauda epididymidis. After perfusion, epididymides were left
overnight in Bouin fixative, dehydrated, and embedded in paraffin.
Longitudinal tissue sections of 6 µm were cut on a microtome and mounted on
glass slides.
LM Immunohistochemistry
Sections were deparaffinized with xylene and rehydrated through a series of
graded alcohol solutions. Endogenous peroxidase activity was neutralized using
70% alcohol containing 1% hydrogen peroxide. After hydration, the sections
were bathed in phosphate-buffered saline containing 300 nM glycine for 5
minutes to block free aldehydes. Immunohistochemical staining of the sections
was done using a Vectastain Elite ABC Kit (Vector Laboratories, Burlingame,
Calif). Rabbit anti-Id1 (1:1400), -Id2 (1: 800), -Id3 (1:1000), and -Id4
(1:200; all from Santa Cruz Biotechnology, Santa Cruz, Calif)
affinity-purified polyclonal antibodies were used. Tissue sections to be
stained for Id1 and Id2 were incubated for 30 minutes (23°C) with normal
goat blocking serum, and sections to be stained for Id3 and Id4 were incubated
for 2 hours in a 5% bovine serum albumin (BSA) and goat serum blocking
solution to block nonspecific binding. Sections to be stained for Id1 and Id2
were incubated in a humidified chamber for 2 hours at 23°C with the
indicated antibodies diluted in the blocking serum. Sections to be stained for
Id3 and Id4 were incubated overnight at 4°C with the indicated antibody in
normal goat serum and 5% BSA. Bound antibodies were observed through the use
of biotinylated goat anti-rabbit immunoglobulin G secondary antibodies and the
avidin–biotinylated horseradish peroxidase complex. Sections were
counterstained with a 0.05% methylene blue solution, dehydrated in ethanol and
xylene, and mounted with cover slips using Permount. Negative controls were
treated the same way, except that the primary antibodies were preincubated
with the corresponding peptides mapping to the carboxy terminus of each
protein (Id1, Id2, Id3, and Id4; all from Santa Cruz Biotechnology). The
immunohistochemical results were analyzed using a LM to determine the
localization of Id1, Id2, Id3, and Id4 proteins in the epididymis. Eighteen to
21 sections per group were prepared and examined.
RNA Isolation and Reverse Transcription Reaction
Epididymides from adult rats were collected; divided into initial segments,
caput, corpus, and cauda regions; and immediately frozen in liquid nitrogen.
Tissues were stored at -80°C until used for RNA extraction. Epididymal
regions were crushed under liquid nitrogen, and RNA was extracted and DNase
treated using the RNeasy Midi kit (Qiagen, Mississauga, Canada), according to
the manufacturer's instructions. The concentration of RNA was measured by
spectrophotometric ultraviolet analysis (DU7 spectrophotometer; Beckman,
Montréal, Canada). From each sample, 5 µg of RNA was run on a 1%
agarose gel to assess the quality of the sample. First-strand complementary
DNA was synthesized from total RNA using SuperScript II (Invitrogen, Carlsbad,
Calif) with oligo (dT)12–18 primer (Invitrogen). For each
sample, a no-reverse transcription (RT) control was done in which all
conditions were identical, minus the Superscript II enzyme. This verified the
absence of genomic DNA in polymerase chain reactions (PCRs).
SYBR Green Quantitative Real-Time PCR
Oligonucleotide primers (sequences shown in the
Table) were designed using
Primer3
(http://frodo.wi.mit.edu/)
to previously described sequences for Id1, Id2, Id3, Id4, and cyclophilin.
Primers were synthesized by the Sheldon Biotechnology Centre (McGill
University, Montréal, Canada). For quantitative PCR, a real-time
fluorometric thermocycler was used (LightCycler; Roche Diagnostics, Laval,
Canada). Each group consisted of 4 separate samples, and each sample was
measured in duplicate. All genes were normalized against cyclophilin, a
housekeeping gene for which mRNA expression does not change across epididymal
regions (Palladino and Hinton,
1994). The PCR Mastermix (20 µL) contained 2x QuantiTect
SYBR Green PCR Master Mix (Qiagen), with 2.5 mM MgCl2, 0.5 µM
forward primer, 0.5 µM reverse primer, and 1 µL cDNA from the RT
reaction. PCR was done as follows: initial denaturation at 95°C for 15
minutes, followed by 60 cycles of denaturation at 95°C for 10 seconds,
annealing at 54°C–60°C for 5 seconds
(Table), and extension at
72°C for 20 seconds. Finally, a melting curve analysis was conducted by
continuous fluorescence acquisition during temperature elevation from 65°C
to 95°C at 0.2°C/s to verify the absence of genomic DNA and
artefactual primerdimers. In each reaction, a standard curve was prepared from
serial dilutions of a standard containing equal amounts of cDNA from all
epididymal regions.
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Statistical Analysis
All statistical analyses were done using 1-way analysis of variance and
multiple comparison testing using the Holm-Sidak post hoc test. Data are
presented as means plus or minus standard error of the mean. For all analyses,
the level of significance was set at P < .05.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Immunocytochemical Analysis for Id1
Immunocytochemistry was done to complement the PCR data and to assess the
cellular distribution of Id proteins along the epididymis. Id1 protein was
found to be expressed in all 4 regions of the epididymis
(Figure 2a through d) and was
primarily localized to myoid cells surrounding the epididymal epithelium.
Consistently low–relative intensity immunoreactivity was found in the
initial segment and caput epididymidis
(Figure 2a, b, and f). The
corpus epididymidis showed a slightly more intense reaction
(Figure 2c and f). Only a thin
band of immunoreactive myoid cells was observed in these 3 proximal regions of
the epididymis. An intense reaction was observed in the cauda epididymidis,
where immunostaining was observed in a significantly greater number of myoid
cells and was of much greater intensity
(Figure 2d and f). Thick bands
of immunoreactive myoid cells are seen in this most distal region of the
epididymis. Preincubation of the primary antibody with the Id1 peptide
completely abrogated immunoreactivity
(Figure 2e).
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Immunocytochemical Analysis for Id3
Id3 immunoreactivity was observed in the 4 epididymal regions. However,
unlike the other Id proteins, staining was primarily confined to the nuclei of
principal cells along the entire tissue
(Figure 4). Light staining of
basal cells was also observed, and myoid cells were immunoreactive
predominantly in the cauda epididymidis. The initial segment of the epididymis
showed relatively low-intensity immunoreactivity for Id3, which was localized
to the nuclei of principal cells, basal cells, and apical cells
(Figure 4a and f).
Immunoreactivity in the caput epididymidis was primarily localized to the
nuclei of principal cells, but the reaction intensity was less than that of
the initial segment (Figure 4b and
f). The corpus (Figure 4c and
f) and cauda epididymidis showed an increase in intensity of
immunoreactivity in the nuclei of principal cells, and immunoreactivity of
myoid cells was also seen in these regions. The intensity of immunoreactive
principal cells was greater in the cauda epididymidis than in any other
region. In addition, a large number of highly immunoreactive myoid cells was
present in this region (Figure 4d and
f). Control sections showed no staining over the epithelium
(Figure 4e).
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Immunocytochemical Analysis for Id4
The cellular distribution of Id4 was observed to be very similar to that of
Id1 with one exception; in addition to immunoreactive myoid cells, narrow
cells were also reactive in the initial segment
(Figure 5a and f). In the
caput, corpus, and cauda epididymidis, Id4 immunostaining was found to be
restricted to myoid cells surrounding the epididymal epithelium
(Figure 5b through d and f). The most intense reaction was observed in the cauda epididymidis, where the
greatest number of myoid cells is found. As was the case for the other Ids,
preincubation of the primary antibody with the Id4 peptide abrogated
immunoreactivity (Figure
5e).
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In Silico
Using the NCBI rat genomic Blast search program, we looked for the presence
of androgen response elements (ARE) in the 3-kb upstream region of the 4 Id
genes. The ARE consensus sequence is a 15-nucleotide–long imperfect
palindromic sequence consisting of two 6-bp half-sites that are separated by a
3-nucleotide–long spacer. Natural AREs often include one virtually
canonical half-site (TGTTCT) and the other half-site of considerable deviation
from this sequence. Near-consensus ARE sequences that match at least 9 of 12
nucleotides have been found in the regulatory regions of a number of
androgen-responsive genes (Nelson et al,
2002). We have identified a potential ARE located at -876- to
-862-bp upstream of the transcriptional start site of the Id3 gene
(AATAAAtagTGTTCT); this sequence differs from the ARE consensus sequence by
only 3 bp, which are located in the -2, -5, and -6 positions. Interestingly,
potential ARE sequences were found in the -721- to -707-bp upstream region of
the human Id3 gene and in the -705- to -691-bp upstream region of the mouse
Id3 gene, and these were both identical to the one observed in the rat. A
functional analysis of this sequence was not done. The 3-kb upstream regions
of the rat Id1, Id2, and Id4 genes were devoid of potential ARE sequences.
Effect of Orchidectomy on Id3 Expression Along the Epididymis
Because of the presence of this potential ARE sequence upstream of the Id3
gene, we determined the effect of orchidectomy on Id3 protein expression
throughout the length of the epididymis. We looked at protein expression
because this gave us information on cellular localization as well as
expression levels. Postorchidectomy, Id3 immunostaining was localized to the
same cell types and was observed in the same proportions as in control
tissues. At 9 days postorchidectomy, when all testicular input has been
removed from the luminal fluid, immunoreactivity increased significantly in
the initial segment, where there is intense staining in the nuclei of
principal cells; decreased slightly in the caput and corpus epididymidis; and
showed an even greater decrease in the cauda
(Figure 6a through d). At 35
days after orchidectomy, immunoreactivity in the initial segment was almost
completely abrogated (Figure
6e). Both the number of principal cell nuclei and the intensity of
staining were slightly increased in the caput epididymidis, while these values
remained the same in the corpus epididymidis
(Figure 6f and g). In the cauda
epididymidis, principal cell staining decreased, while myoid cell
immunoreactivity increased (Figure
6h). Thus, expression of Id3 is differentially regulated along the
duct when androgens and/or testicular factors are removed.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The presence of several Id proteins in a tissue could reflect redundancy in function or different activities for these proteins. Evidence for the latter is that outside the highly conserved HLH domain, the Id proteins display extensive sequence divergence (Norton et al, 1998). Further evidence of this nonredundancy in the epididymis is our observation that Ids are expressed in different constellations of cell types, indicating distinct regulatory functions for these HLH proteins: Id1 was found to be localized primarily to myoid cells, Id2 to clear cells, Id3 to principal cells, and Id4 to myoid and narrow cells.
The molecular basis for the region-specific differences in gene expression in the epididymis is not yet resolved. Ids are differentially expressed along the duct. This is interesting, particularly in the context of the mechanism of action of Ids, which is to sequester and functionally antagonize other transcriptional regulators. This, combined with the opposing expression profiles of many other differentially expressed genes in the epididymis (Jervis and Robaire, 2001), indicates that members of the Id family of transcription factors may play an important role in the regulation of gene expression. Ids may be components that are at the top of a hypothetical transcriptional cascade; that is, they may control a cascade of transcription factors that coordinate a gene expression program.
The expression of Id1, Id2, Id3, and Id4 has been positively correlated with proliferation in several epithelial cell types (ie, they often are up-regulated in undifferentiated, proliferating cells and down-regulated at the start of cellular differentiation). In tissue culture models, ectopic expression of Id proteins can result in a block in differentiation processes and cell immortalization. For instance, ectopic expression of Id1 in Schwann cells was shown to strongly repress myelin gene promoter activity (Thatikunta et al, 1999). Although Id proteins have traditionally been viewed exclusively as negative regulators of cellular differentiation (Jen et al, 1992; Shoji et al, 1994; Lister et al, 1995), their function may not be so 1-dimensional. Recent studies have revealed possible molecular promiscuity and much wider biological roles for this family of regulatory proteins. For instance, it has been shown that all Ids are expressed in postmitotic, terminally differentiated Sertoli cells (Chaudhary et al, 2001); Id2 protein may actually promote differentiation in response to follicle stimulating hormone in these cells. Id2 protein expression is higher in adult alveolar epithelial cells than in the corresponding fetal cells; therefore, Id2 is thought to be required for the determination and maintenance of these cells (Liu et al, 2000). Immunohistochemical localization of Id proteins in the small intestine showed that the highest concentrations of Ids are found in fully differentiated, nonproliferating cells (Wice and Gordon, 1998). Further evidence of the more complex functions of Id proteins lies in a study done on pancreatic beta cells, which indicated that Id proteins promote, rather than inhibit, insulin gene transcription (Wice et al, 2001).
Id1, Id2, Id3, and Id4 proteins are regulated by a plethora of growth factors and mitogens in many different cell types (Brennan et al, 1991; Christy et al, 1991; Biggs et al, 1992; Deed et al, 1993; Hara et al, 1994). We have found a potential ARE located -876 to -862 bp upstream of the transcriptional start site of the Id3 gene. The ARE consensus sequence is recognized by the androgen receptor as well as the glucocorticoid receptor and the progesterone receptor. Interestingly, it is clear that principal cells in the rat epididymis stain heavily for androgen receptors and that intracellular localization is specifically nuclear. Principal cells are also particularly sensitive to the removal of circulating androgens, whereas other cell types appear to be unaffected (Orgebin-Crist and Davies, 1974; Moore and Bedford, 1979; Ezer and Robaire, 2003). Id3 was the main protein of interest because of its localization to principal cells, which outnumber all other cell types combined by at least a threefold measure, and principal cells are the main cell type involved in secretion of luminal components (Robaire and Hermo, 1988). A recent study done in our laboratory indicates that Id3 transcript levels may be androgen regulated in the epididymis (Ezer and Robaire, 2003). Therefore, perhaps the presence of this presumptive ARE is the reason why Id3 is expressed to a greater degree than the other Ids in principal cells along the duct. Our observation that Id3 is differentially expressed at the protein level along the epididymis in response to orchidectomy is of interest because these region-specific changes are consistent with what was seen at the mRNA level in the study by Ezer and Robaire (2003). Orchidectomy results in region-specific changes in Id3 expression that follow different patterns with respect to time postorchidectomy: transient up-regulated expression in the initial segment, early declining expression in the caput and corpus epididymidis, and progressively declining expression in the nuclei of principal cells in the cauda epididymidis. The initial segment shows a marked increase in immunoreactivity at 9 days postorchidectomy, whereas expression is significantly decreased in the cauda epididymidis. This may be a result of androgen withdrawal itself or a result of the removal of testicular factors. The novel pattern of expression of this inhibitor of transcription factors provides new molecular insight into the regulation and differential expression of genes, the protein products of which are secreted by principal cells along the duct. We speculate that this may lead to the region-specific differences in gene and protein expression that are characteristic of the epididymis.
Individual Id proteins have different affinities for various transcriptional regulators; that is, each Id protein has a distinct fingerprint of preferred partners (Langlands et al, 1997). Id proteins can sequester both "positive" and "negative" transcription factors, and the net biological effect is the sum of these interactions to either promote or inhibit gene transcription.
In this study, we used real-time PCR and immunohistochemical analysis to characterize the expression, in the epididymis, of 4 transcription factors belonging to the HLH family of proteins: Id1, Id2, Id3, and Id4. We also identified a potential ARE within 1-kb upstream of the transcriptional start site of the Id3 gene, and looked at Id3 protein expression along the duct in orchidectomized and in aged animals. The current observations establish novel patterns of expression for Ids in the rat epididymis; these could potentially act as regulators of many downstream genes associated with the region-specific differences that are the hallmark of this tissue.
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Footnotes |
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