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From the Departments of * Urology and
Cell Biology, University of Virginia School of
Medicine, Charlottesville, Virginia.
| Correspondence to: Dr Terry T. Turner, Department of Urology, University of Virginia School of Medicine, PO Box 800422, Charlottesville, VA 22903 (e-mail: ttt{at}virginia.edu). |
| Received for publication September 22, 2003; accepted for publication December 8, 2003. |
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Abstract |
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Key words: Gene transcription, efferent duct ligation
Hedgehog (hh) proteins also transmit patterning information in the embryo (Wallis and Muenke, 1999; Nybakken and Perrimon, 2002). This family of genes has evolved from the hh gene first identified in Drosophila as a segment polarity or patterning gene (see Perrimon, 1995), but in mammals, there are 3 forms of the gene: Indian (Ihh), desert (Dhh), and sonic (Shh). In tissues important to reproduction, Ihh is involved in mammary gland development and maintenance (Lewis et al, 1999) and in the uterine endometrium during implantation (Takamoto et al, 2002). Dhh is produced in the testis, specifically in Sertoli cells, where it plays a role in germ cell development (Bitgood et al, 1996). Shh is active during the development of the prostate (Podlasek et al, 1999) and external genitalia (Perriton et al, 2002). A role for Shh in adult tissues has yet to be described.
Like all members of the hh family, Shh signals through a multistep pathway. Briefly, Shh is typically a protein synthesized and secreted by epithelial cells, and its receptor, patched (Ptc), spans the membrane of its mesenchymal target cells (Burrows, 2000; Walterhouse et al, 2003). Shh binding relieves the Ptc inhibition of a G protein–coupled membrane protein, smoothened (Smo). Smo, through a signaling process involving protein kinase A, another kinase, fused (Fu), and its inhibitor, suppressor of fused (SuFu), eventually activates the Gli (glioma-associated oncogene) protein(s) Gli-1, Gli-2, or Gli-3 for translocation to the nucleus and regulation of target gene transcription (Lamm et al, 2002; Nybakken and Perrimon, 2002; Walterhouse et al, 2003). Major components of the Shh signaling pathway have been localized in the urogenital sinus and developing prostate, where Shh signaling and Shh gene expression are at least partially androgen-dependent (Podlasek et al, 1999; Barnett et al, 2002) and where cyclopamine inhibition of Shh signaling inhibits prostatic bud development in vitro (Lamm et al, 2002).
The role of Shh in patterning, its importance in the developing urogenital tract, and the highly patterned function of the adult epididymis raised the question of whether Shh has a continued role in that adult organ. In the present study, we have determined whether major elements of the Shh signaling pathway are present in the adult mouse epididymis at the gene and protein level.
Additionally, previous evidence in the rat (Nicander et al, 1983; Fan and Robaire, 1998; Turner and Riley, 1999) and mouse (Abe et al, 1982) suggests that lumicrine factors are important in the maintenance of the epididymal epithelium. This is especially true in the most proximal segments of the epididymis, where it has been shown in the rat that the absence of lumicrine factors induces epithelial apoptosis in a segment-specific manner (Turner and Riley, 1999; Turner et al, 2003). In the present study, we have also determined whether the structural segmentations of the adult mouse epididymis are maintained if lumicrine factors are chronically absent from the testis, and we have determined whether the expression of Shh pathway molecules in the epididymis is altered under those same conditions.
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Materials and Methods |
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Efferent Duct Ligation
Unilateral efferent duct ligation (EDL) was performed on adult male mice in
a conventional manner as described previously for the rat
(Turner and Riley, 1999).
Epididymides having different durations of EDL (see below) and companion,
sham-operated, control epididymides were collected from mice killed by
anesthetized cervical dislocation. Epididymides were immediately removed and
placed in ice-cold phosphate-buffered saline. Epididymal fat was quickly
removed, and the tissues were either processed intact for histology (see
below), or they were separated into caput, corpus, and cauda regions,
flash-frozen in liquid N2, and either stored at -80° or used
immediately for RNA or protein extraction.
Histology
Tissues were collected for morphological evaluation at 0 (control), 6, 12,
24, 48, 72, and 720 hours after EDL. Epididymides were pegged flat on thin
platforms of dental wax and fixed in Bouin's solution, paraffin embedded, and
stained with hematoxylin and eosin. These tissues were subjected to
longitudinal, whole-epididymis sectioning and were used for 1) measurement of
lumen diameter and epithelial height in segments 1, 2, and 3 of the caput
epididymis (see Turner et al,
2003, for illustration); 2) determination of epithelial apoptosis
in segments 1, 2, and 3; and 3) evaluation of the maintenance of distinct
segments throughout the epididymis as noted by the presence of connective
tissue septa (CTS).
Determination of lumen diameter and epithelial height was performed by use of a micrometer reticle on a light microscope (Carl Zeiss, Thornwood, NY). Five tubules per segment in each epididymis were measured using only those tubule profiles that were circular in appearance. Epithelial apoptosis was evaluated 24 hours after EDL in segments 1, 2, and 3 by use of the Apostain (Alexis Corp, San Diego, Calif) technique as previously described by our laboratory (Lysiak et al, 2001). The apoptotic nuclei per tubule cross section in segments 1, 2, and 3 were determined using only tubule profiles appearing circular in cross section, with 5 tubules per segment from 5 epididymides, each, representing control and 24-hour EDL tissues. All of the above studies focused on segments 1, 2, and 3 of the mouse caput, because previous evidence in the rat (Turner and Riley, 1999) and preliminary evidence in the mouse suggested the most prominent effects were exhibited in those proximal segments. Determination of the presence and location of CTSs within each epididymal region was performed as described previously (Turner et al, 2003) and was performed at each of the 7 time points (0–720 hours after EDL) described earlier.
Relative Quantitative Reverse Transcriptase-Polymerase Chain Reaction
Five tissues per caput, corpus, and cauda epididymal regions were
snap-frozen, pooled, and homogenized in liquid nitrogen. Homogenate RNA was
extracted (TRIzol; Invitrogen Life Technologies, Carlsbad, Calif) according to
the manufacturer's instructions and treated with DNase I (Invitrogen) to
eliminate DNA contamination. In some cases, total testis RNA and prostate RNA
were also collected using the same techniques. One microgram of total RNA was
reverse transcribed (RT) to obtain complementary DNA (cDNA) using Superscript
II reverse transcriptase and random hexamers (Invitrogen) according to the
manufacturer's instructions. The polymerase chain reaction (PCR) was performed
in a gradient thermal cycler (Biometra, Göttingen, Germany) using
Platinum High Fidelity Taq Polymerase (Invitrogen) and specific
forward and reverse primers for Shh, Ptc1, and Gli1 as
specified in the Table.
Ptc1 is the most prominently expressed form of the Ptc gene
in developing testis and prostate
(Walterhouse et al, 2003); thus, it was the gene selected for study. Gli1 has previously been
demonstrated to be expressed in the mesenchyme of the developing prostate
(Lamm et al, 2002), is known to
be expressed in hh-responsive cells, and is the most prominent family member
acting as a direct transcription factor
(Goodrich and Scott, 1998; Walterhouse et al, 1999).
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Primers and competimers for 18S ribosomal RNA (rRNA) (489 bp; Ambion, Austin, Tex) were added after pilot experiments determined the appropriate ratios of competimers to primers to use with the amplification of each PCR product. All reactions had a denaturing temperature of 94°C and an extension temperature of 68°C in addition to the annealing temperature shown in the Table. All RT-PCR products were separated on 1% agarose gels. Images were captured using a ChemImager system (Imogen Technologies, Alexandria, Va), and band densities were determined using ImageQuant software (Molecular Dynamics, Sunnyvale, Calif). The band density of each RT-PCR product of interest was recorded relative to the density of the 18S product. From 5 to 6 different RNA samples (5 tissues pooled per sample) were subjected to RT-PCR for each of the products of interest at 0, 1, 3, 10, and 15 days after EDL.
The identity of each of the 3 amplified products of interest was verified by sequence analysis (Molecular Core Facility at the University of Virginia, Charlottesville, Va) after cloning into pCR BluntII-TOPO vectors (Invitrogen).
Western Blot Analysis
Tissues (testis, prostate, and caput, corpus, and cauda epididymides) were
collected from animals at the same times after EDL as described above and were
snap-frozen and homogenized as described above. After evaporation of the
liquid nitrogen, buffer (50 mmol of Tris per liter, 150 mmol of NaCl per
liter, 10 mmol of EDTA per liter, 1.0% NP-40, 0.5% sodium deoxycholate, and
0.1% sodium dodecyl sulfate) containing protease inhibitors (leupeptin, 100
µmol/L; PMAF, 1 mmol/L; aprotinin, 1 µg/mL; and E-64, 10 µmol/L) was
added to the samples. Samples were placed on ice for 10 minutes before
centrifugation at 12 000 x g for 10 minutes at 0°C. The
supernatant was collected, and the protein concentration was determined with
the BCA (bicinchoninic acid) assay (Pierce, Rockford, Ill) according to the
manufacturer's instructions. Protein samples were diluted in Laemmli buffer
(Laemmli, 1970), boiled for 5 minutes, loaded onto gels at 60 µg per lane,
and electrophoresed on either 8% (Ptc-1 and Gli-1) or 12% (Shh) gels.
Separated proteins were electrotransferred to nitrocellulose membranes
(BioRad, Hercules, Calif). Membranes were then blocked with 5% milk and 0.1%
Tween-20 in Tris-buffered saline, pH 7.4, for 1 hour prior to overnight
incubation with the primary antibody. The antibodies used were a goat
polyclonal antibody against mouse Shh N-terminal peptide (AF464, 1:1000
dilution; R&D Systems, Minneapolis, Minn), a goat polyclonal antibody
against mouse Ptc (G-19, 1:1000 dilution; Santa Cruz Biotechnology, Santa
Cruz, Calif), and a rabbit polyclonal antibody against mouse Gli-1
(100-401-233, 1:10 000 dilution; Rockland Immunochemicals, Gilbertsville, Pa).
Blots were washed and incubated with the appropriate peroxidase-conjugated
secondary antibody. Negative controls were blots that received secondary
antibody only. Immunorecognition was detected using enhanced chemiluminescence
(Pierce). Images were captured and analyzed densitometrically by ImageQuant
software (Molecular Dynamics). Equal protein loading of gel lanes was ensured
by staining the nitrocellulose blots with Ponceau S (Sigma Chemical Co, St
Louis, Mo).
Immunohistochemical Localization
The antibodies against Shh, Ptc-1, and Gli-1 listed above were also used
for immunohistochemical localization of proteins in control tissues. Primary
antibody dilutions for Shh, Ptc-1, and Gli-1 were 1:100, 1:500, and 1:500,
respectively. Immunohistochemistry was carried out with conventional
techniques as described previously for the laboratory
(Bomgardner et al, 2003) on
tissues that had been fixed in 4% paraformaldehyde, paraffin embedded, and
counterstained with hematoxylin. Biotinylated secondary antibodies were
visualized with avidin-biotin complex (Elite ABC Kit; Vector Labs, Burlingame,
Calif) and diaminobenzidine (Sigma). Negative controls were tissues carried
through the procedure with no exposure to the primary antibody. Also, blocking
peptides for Shh and Ptc-1 were available and used at 5x and 10x
the concentration of the antibody to evaluate the specificity of the primary
antibody.
Statistical Evaluation
Numerical data in multiple-group comparisons were analyzed by analysis of
variance followed by the Tukey range test (P < .05).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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EDL in the mouse causes a clearance of sperm and, presumably, other testicular contribution to the epididymal lumen in a pattern that is also illustrated in Figure 1A. For example, segment 1 is typically cleared of sperm within 6 hours of EDL, and segment 3 is cleared within 24 hours; however, even at 30 days (720 hours) after EDL, some sperm are still retained in the extreme distal cauda. The number of CTSs segmenting the mouse epididymis was not significantly altered at any time after EDL. As an example, the number of CTSs in the caput, corpus, and cauda epididymides of mice at the most extended time after EDL, 30 days, was 5.4 ± 0.5, 1.7 ± 0.3, and 3.0 ± 0.9, respectively.
The mean values (mean ± SE) for epithelial height in segments 1, 2, and 3 of the sham-operated, control epididymides ("0 hr" after EDL values in Figure 1B) were 46.5 ± 0.4 µm, 26.0 ± 0.2 µm, and 24.6 ± 0.3 µm, respectively, and the values were all significantly different from each other (Figure 1B). The epithelial height in segment 1 became significantly less than the controls at 12 hours after EDL and remained reduced for the duration of the study. The epithelial height in segment 2 became significantly greater than controls at 24 hours after EDL, remained that way until 72 hours after EDL, but had returned to control values by 30 days after EDL. The epithelial height in segment 3 fluctuated but remained significantly elevated after 72 hours following EDL (Figure 1B).
Lumen diameters (mean ± SEM) in segments 1, 2, and 3 of control epididymides were 58.1 ± 0.9 µm, 49.6 ± 0.8 µm, and 53.0 ± 0.9 µm, respectively, with segment 1 values being significantly greater than those for segments 2 and 3, and segment 3 values being significantly greater than those for segment 2 (Figure 1C). Lumen diameters in segment 1 began a trend downward within 12 hours of EDL but did not become significantly reduced until 48 hours after EDL (Figure 1C). Segment 2 lumen diameters became significantly reduced within 6 hours after EDL, and segment 3 diameters became significantly reduced within 24 hours after EDL.
Epithelial apoptosis was not detected by Apostain in segment 1, 2, or 3 of the control epididymides, but it was detected in segment 1 of the epididymides 24 hours after EDL. The number of apoptotic nuclei per tubule cross section detected in segments 1, 2, and 3 of the mouse epididymis 24 hours after EDL was 6.6 ± 0.9, 0.1 ± 0.0, and 0.0 ± 0.0 ± 0.0, respectively. Thus, 24 hours after EDL, loss of testicular contribution induced apoptosis in segment 1, specifically.
Sonic Hedgehog Pathway in the Adult Mouse Male Tract: Gene and Protein Expression in Different Tissues
The 3 hh pathway genes studied were all expressed in adult male tract
tissues at both the gene and protein level, but expression levels differed
depending on the gene, protein, and tissue being analyzed. Shh
expression was not detected by semiquantitative RT-PCR in the mouse testis but
was detected in the epididymis, with a trend of increasing intensity in the
distal regions of the organ (Figure
2A). Shh detection in the adult mouse prostate was either
absent or extremely faint (Figure
2A) in the samples assayed. The absence of Shh gene
expression in the adult testis and prostate was matched by the absence of Shh
protein in those tissues, and the presence of Shh gene expression in
the epididymis was generally correlated with Shh protein presence
(Figure 2A). Both the gene and
protein appeared to increase distally
(Figure 2). Here and elsewhere,
Ponceau S staining of electroblot membranes verified equivalent protein
loading (data not shown).
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Ptc1 gene expression was detected in all 3 adult tissues—testis, epididymis, and prostate (Figure 2B). There was no consistent pattern of difference in Ptc1 expression in the 3 epididymal regions (Figure 2B). Also, Ptc-1 protein was detected by Western blot analysis in the extracts of all 3 reproductive tract tissues (Figure 2B). The pattern of protein presence within the epididymal regions was variable, but Figure 2B illustrates the most common pattern (ie, diminished detection of Ptc-1 in the corpus).
Gli1 expression was detected in the testis, epididymis, and prostate (Figure 2C). Epididymal expression of Gli1 typically appeared more prominent than in the testis or prostate, but there was no consistent pattern of Gli1 gene expression among the epididymal regions sampled. Gli-1 protein was consistently detected in protein extracts from the testis, epididymis, and prostate (Figure 2C). Within the epididymis, there was always diminished band intensity in the corpus epididymidis and, in some cases, it was absent (Figure 2C).
Shh protein was immunolocalized to the epididymal epithelium (Figure 3). This was true throughout the epididymis, though only the proximal caput (Figure 3A through C) and mid-cauda (Figure 3D through F) are illustrated. Immunostaining in the presence of the primary antibody (Figure 3D and E for caput and cauda, respectively) was markedly diminished in the presence of blocking peptide (Figure 3C and F), though background staining in the epithelium was still existent. Attempts to reliably immunolocalize Ptc-1 and Gli-1 were not successful. The Ptc-1 blocking peptide bound nonspecifically to the tissue sections even in the absence of the primary antibody, and immunostaining for Gli-1 was not sufficiently localized to be interpretable in the absence of a blocking peptide.
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The Effect of EDL on the Shh Pathway in the Epididymis
Shh expression remained evident in all regions of the adult mouse
epididymis after EDL (Figure
4A). Shh expression in the caput epididymis increased
significantly by 10 days after EDL and then returned to control levels by 15
days (Figure 4B; n = 6). The
corpus epididymidis exhibited a significant change in the expression level 15
days after EDL, but the cauda showed no significant difference in Shh
expression during the experimental period
(Figure 4B).
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Ptc1 expression also continued in all regions of the adult epididymis after EDL (Figure 5A). Caput and corpus expression of the Ptc1 gene increased significantly by 15 days after EDL, but the cauda epididymidis never exhibited a significant change in the gene's expression (Figure 4B; n = 5).
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Gli1 expression continued in all 3 regions of the adult mouse epididymis after EDL (Figure 6A), but expression was variable both within and between times after the operation (Figure 6B). Only the caput epididymidis demonstrated a significant change in Gli1 expression, which began by 3 days after EDL and was again detected 15 days after EDL (Figure 6B).
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Discussion |
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In fact, maintenance of the CTSs in the mouse epididymis does not require lumicrine factors, at least during a 30-day period, and changes in Shh, Ptc1, and Gli1 expression after EDL are not associated with changes in the organ's segmentation. Even a major disruption like the induction of apoptosis in segment 1 of the epididymis 24 hours after EDL (see "Results"), which was associated with a contemporaneous, significant loss of epithelial height (Figure 1B), was not accompanied by a change in Shh pathway gene expression in the caput (Figures 4, 5, 6).
The second and third hypotheses of these experiments proved to be correct. The 3 Shh pathway molecules studied were present at both the gene and protein level; however, neither the regional examination for the 3 genes and proteins (Figure 2) nor the immunohistochemical localization of Shh (Figure 3) indicated a strict segmentation of Shh signaling in the epididymis. Any role that the pathway plays in segmental function of the epididymal epithelium is likely to be as one among a number of factors influencing tubule structure and function. The localization of Shh protein to the epididymal epithelium throughout the duct is consistent with its conventional role in epithelial-mesenchymal signaling as studied during development. This suggests the possibility that this same type of interaction occurs in this adult tissue, but this remains to be established and to be differentiated from the possibility that Shh is secreted into the lumen for signaling to the more distal epithelium or to the luminal spermatozoa. The localization of Ptc-1, the Shh receptor, did not prove possible in the present experiments but will be important in determining those latter possibilities.
Shh is present in the prostate (Podlasek et al, 1999) and male genitalia (Perriton et al, 2002) during development, but its presence in the epididymis has not been documented. Bitgood et al (1996) mentioned without documentation that Shh expression occurs in the fetal mouse epididymis, and experiments currently under way in another laboratory provide preliminary indications that this is correct (Hinton and Bushman, unpublished data). Nevertheless, to our knowledge, the present report is the first to show both Shh gene expression and protein presence in the organ, especially in the adult, and their absence in adult testis and prostate.
Shh protein presence in a tissue is meaningless in a tissue unless its membrane receptor, Ptc, and Gli transcription factor(s) are also present. Both Ptc1 and Gli1 gene expression and protein presence were detected in testis, epididymis, and prostate (Figure 2B and C). Ptc1, the gene for the Shh receptor, has previously been shown to be expressed in the mouse testis (Bitgood et al, 1996), but, to our knowledge, the present work is the first to determine either Ptc1 or Gli1 gene expression or protein presence in the epididymis, whether fetal or adult. Together, these results imply that the Shh signaling pathway is present and operative in the adult epididymis but not in adult testis or prostate due to the absence of Shh protein.
Shh, Ptc1, and Gli1 expressions in the epididymis were all significantly altered at various points after deprivation of lumicrine factors by EDL. It should be noted that band intensities reflecting gene expression in the "A" panels of Figures 4, 5, 6 often appear not to match the mean data reflected in the "B" panels. This is because the data in the histograms are the mean of ratios of the specific genes to 18S messenger RNA (mRNA) in their same lanes, not to the direct gene product band density from the gels in the illustration. Further, the intraepididymal expressions of individual genes did vary from sample to sample; thus, no individual gel matches the mean data at all points. Individual sample variability is why the mean data from a minimum of 5 samples were analyzed. Shh expression in the caput and corpus was significantly and sequentially increased, first in the corpus at 10 days after EDL and then in the cauda 15 days after EDL (Figure 4). In both regions, this time of change in gene expression preceded the complete clearance of sperm (marker for remaining testicular contribution) from the region (Figure 1A). In the distal regions of the epididymis, the decreases in the tubule lumen diameter and the loss of the apparent density of the remaining sperm in the lumina are also signals that testicular contribution is being reduced, but they occur before the complete clearance of sperm. By these empiric evaluations, retrospectively, the changes in Shh expression in the corpus and cauda at 10 and 15 days after EDL, respectively, were consistent with major, though not complete, loss of luminal testicular products. The rise in Shh expression seen at 10 days after EDL, and then the increase in the cauda at 15 days after EDL (Figure 4), suggests a wave of increased Shh expression passing down the epididymis due to the loss of some lumicrine signal.
Ptc1 expression significantly increased in both the caput and corpus 15 days after EDL (Figure 5). In the corpus, this increase was consistent with the increased expression of Shh at 15 days after EDL (Figure 4), but in the caput, this was not the case.
In summary, the Shh pathway is present in the adult epididymis (Figure 1A through C). Qualitative evidence (Figure 2A) indicates that Shh, the initiator of the signaling cascade, tends to increase distally, and it is the distal Shh and Ptc1 gene expressions that are altered after chronic EDL (Figures 4 and 5). This implies that Shh and Ptc1 gene expressions in those regions are regulated in a manner influenced either directly or indirectly by luminal content. Genes that are regulated by their local environment potentially serve specific functions, and this possibility remains of interest. Subsequent experiments will determine the effects of Shh pathway inhibition on selected features of adult epididymal function.
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Acknowledgments |
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Footnotes |
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160 kd), Ptc (
