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BOR - Papers in Press, published online ahead of print May 14, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.013508
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BIOLOGY OF REPRODUCTION 69, 816–827 (2003)
DOI: 10.1095/biolreprod.102.013508
© 2003 by the Society for the Study of Reproduction, Inc.

Male Reproductive Tract

Altered Sonic Hedgehog Signaling Is Associated with Morphological Abnormalities in the Penis of the BB/WOR Diabetic Rat1

Carol A. Podlasek2,3, David J. Zelner3, Joseph D. Harris3, Cynthia L. Meroz4, Yi Tang4, Kevin E. McKenna4, and Kevin T. McVary3

Departments of Urology3 and Physiology,4 Northwestern University Medical School, Chicago, Illinois 60611


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Erectile dysfunction (ED) is a common and debilitating pathological development that affects up to 75% of diabetic males. Neural stimulation is a crucial aspect of the normal erection process. Nerve injury causes ED and disrupts signaling of the Sonic hedgehog (Shh) cascade in the smooth muscle of the corpora cavernosa. Shh and targets of its signaling establish normal corpora cavernosal morphology during postnatal differentiation of the penis and regulate homeostasis in the adult. Interruption of the Shh cascade in the smooth muscle of the corpora cavernosa results in extensive changes in corpora cavernosal morphology that lead to ED. Our hypothesis is that the neuropathy observed in diabetics causes morphological changes in the corpora cavernosa of the penis that result in ED. Disruption of the Shh cascade may be involved in this process. We tested this hypothesis by examining morphological changes in the penis, altered gene and protein expression, apoptosis, and bromodeoxyuridine incorporation in the BB/WOR rat model of diabetes. Extensive smooth muscle and endothelial degradation was observed in the corpora cavernosa of diabetic penes. This degradation accompanied profound ED, significantly decreased Shh protein in the smooth muscle of the corpora cavernosa, and increased penile Shh RNA expression in the intact penis (nerves, corpora, and urethra). Localization and expression of Shh targets were also disrupted in the corpora cavernosa. Increasing our understanding of the molecular mechanisms that regulate Shh signaling may provide valuable insight into improving treatment options for diabetic impotence.

apoptosis, male sexual function, penis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Erectile dysfunction (ED) is a serious condition that affects 52% of men between the ages of 40 and 70 years. The incidence of ED increases with age, coronary artery disease, peripheral vascular disease, smoking, dyslipidemia, and diabetes mellitus [1, 2]. Diabetes affects 15 million Americans and is a contributing factor in 50% of ED cases. Men with diabetes experience impotence at an earlier age, and the incidence of ED may range as high as 75% [3]. Current treatment options for ED, including intracorporal administration of prostaglandins and vacuum erection devices, are only partially effective [4], and oral sildenafil treatment has been effective in a minority of patients with insulin-dependent diabetes [5]. The cause of ED remains unknown; however, cavernous artery insufficiency, corporeal venoocclusion dysfunction, smooth muscle dysfunction, and autonomic neuropathy are common in patients with ED. Because angiopathy and neuropathy are common complications of diabetes, any one of these risk factors or a combination thereof may be the major pathophysiological mechanism(s) causing diabetic impotence. We focus on one of these contributing factors, neuropathy, which contributes significantly to the pathophysiology of ED both in humans [6] and in rats [7–9]. Our hypothesis is that neuropathy leads to diabetic impotence by causing morphological changes in the penile corpora cavernosa.

Preliminary published reports have included descriptions of morphological changes in the diabetic penis that include a general impairment in responsiveness of the rat corpora cavernosa [10], impaired smooth muscle relaxation [11], a significant decrease in smooth muscle and endothelium of the corpora cavernosa [12], and an increase in the ratio of apoptotic cells in the erectile tissue [13]. These studies were all performed in rats in which diabetes was induced by streptozotocin administration. The streptozotocin model is used widely as an animal model to investigate complications of diabetes mellitus despite the intrinsic problems associated with streptozotocin injection. The streptozotocin rat model displays both vascular and neural changes associated with diabetes, thus making accurate differentiation of the cause (vascular or neural) of the observed abnormal morphology difficult. The BB/WOR rat model of diabetes is a naturally occurring mutation that closely resembles humans in the development and clinical features of type I diabetes. This rat model is uniquely suited for exploration of morphologic changes in the penis that may be attributed to neuropathy alone because it is characterized by profound neuropathy without the confounding vasculopathy observed in other animal models of diabetes. Vasculopathy, as defined by atherosclerosis and microangiopathy, is absent in the BB/WOR rat [14]. Diabetes in the BB/WOR rat is caused by immune-mediated destruction of insulin-producing pancreatic beta cells [15, 16]. Copulatory behavioral testing revealed that diabetic males are severely impaired, and more than half of the diabetic rats failed to exhibit penile erections [17]. Thus, the BB/WOR rat model of diabetes displays both neuropathy and ED, and examination of morphological changes in this model allowed us to investigate the contribution of neuropathy alone to diabetic impotence.

One mechanism through which penile morphology may be altered is by disruption of a cascade of signaling molecules that establish postnatal corporal cavernosal differentiation and homeostasis of adult structures. The morphogen Sonic hedgehog (Shh) has recently been identified as playing a central role both in embryonic development of the penis [18, 19] and in postnatal differentiation of penile tissues [20]. Shh, bone morphogenetic protein 4 (BMP-4), Patched (Ptc), and Hoxd-13 form part of a regulatory cascade that is essential for postnatal morphogenesis and differentiation of the penis. These genes are abundant after birth and in the adult, and their localization in the nerves of the dorsal nerve bundle, smooth muscle, and endothelial lining of the corpora cavernosal sinusoids (Fig. 1) suggests their involvement in postnatal differentiation of the corpora cavernosa. Shh and BMP-4 proteins play a crucial role in establishing the normal morphology of the corpora cavernosal sinusoids as these structures differentiate after birth [20]. These genes continue to function in the adult, where they maintain the sinusoidal structure of the corpora cavernosa. Disruption of Shh signaling in vivo led to the dedifferentiation of corpora cavernosal tissue such that sinusoids were completely absent [20]. Addition of exogenous BMP-4 peptide resulted in enlarged sinusoidal spaces. Data suggest that Ptc [21], the receptor for Shh, and Hoxd-13 [22] (one of 39 Hox genes that define positional identity along the anterior/posterior axis of developing genitalia) are also important in penile morphogenesis and adult homeostasis [20]. The continuing function of Shh and targets of its signaling in maintaining penile morphology in the adult is significant because disruption of Shh signaling resulted in ED as measured by decreased intracavernosal pressure measurement upon electrical stimulation [20]. The ED apparent after inhibition of Shh function was attributed to alterations in corpora cavernosal morphology, including decreased smooth muscle and endothelium [20]. In these studies, we examined changes in expression and localization of Shh cascade members in a diabetic model of neuropathy and ED. We evaluated correlations between these changes and extensive morphological alterations identified in the corpora cavernosa. Insight obtained in these studies may be used to improve treatment of ED.



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  		FIG. 1. Corpora cavernosal sinusoid of the rat penis. Endothelium, smooth muscle, and blood cells are indicated with arrows. Endothelium lines the sinusoidal space. The endothelial cells contain typically flattened nuclei. Smooth muscle lies adjacent to the endothelium in a matrix of collagen, fibroblasts, and interstitial tissue. Smooth muscle cell nuclei appear much less flattened


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Model

BB/WOR diabetic rats were obtained from an established breeding colony (Biomedical Research Models, Worcester, MA). Two types of BB/WOR rats were used: diabetes prone (diabetic) BB/WOR and diabetes resistant (control) BB/WOR rats. The presence of diabetes was determined by examining blood glucose levels (Biomedical Research Models). The onset of diabetes occurs between 60 and 120 days after birth (mean, 96 days) in these rats. The animals were killed for tissue collection 160–190 days after birth after approximately 70–100 days of diabetes expression. Control rats were age matched. Penes were harvested from killed males by sharp dissection (scalpel and scissors) and were either frozen in liquid nitrogen or fixed in 4% paraformaldehyde. In some of the rats, the corpora cavernosa was isolated separately and frozen after removing the urethra and dorsal nerve bundle by dissection under a KAPS dissecting microscope. Animals were cared for in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.

RNA Isolation and Quantification of Gene Expression by Reverse Transcription Polymerase Chain Reaction

Total RNA was extracted separately from intact penes (corpora cavernosa, urethra, and nerve bundle) and pelvic ganglia of BB/WOR diabetic and control rats using the TRIzol (Life Technologies, Gaithersburg, MD) method. Samples were treated with DNase (Promega, Madison, WI) to eliminate genomic DNA contamination. Primers (Table 1) were synthesized at the Northwestern University Biotechnology Facility. Reverse transcription polymerase chain reaction (RT-PCR) was performed using the Gene Amp RNA PCR Core kit (Perkin-Elmer, Branchburg, NJ), and products were restriction digested to confirm that bands represented the sequences of interest. Semiquantitative RT-PCR was performed as described previously [8, 23], using noncompetitive methodology and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or ribosomal protein L-32 (RPL-32) as an endogenous internal standard. All measurements were made in the linear range for Shh and GAPDH (n = 8 controls and 9 diabetics), Ptc and GAPDH (n = 4 controls and 6 diabetics), BMP-4 and RPL-32 (n = 3 controls and 4 diabetics), and Hoxd-13 and GAPDH (n = 5 controls and 7 diabetics). Assays were performed in triplicate on individual tissue specimens, the results were averaged, and the product ratios are reported as the mean ± SEM. The data for each gene were normalized to 1 (maximum value set equal to 1) to present results in a comparable manner. Excel (Microsoft, Redmond, WA) was used for statistical analysis, and a t-test was used to determine significant changes in gene expression.


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  		TABLE 1. Primers used for RT-PCR

Immunohistochemical Analysis

Immunohistochemical analysis (IHC) was performed as previously outlined [8] on control (n = 5) and diabetic (n = 5) rat penes. Sections were incubated with one of the following antibodies: goat polyclonal IgG Shh, Ptc, Bmp-4, and platelet endothelial cell adhesion molecule 1, also known as CD31 (Santa Cruz Biotechnology, Santa Cruz, CA; 200 µg/ml, catalog nos. sc-1194, sc-6149, and sc-6896, respectively) or alpha smooth muscle actin (Sigma, St. Louis, MO). The specificities of Shh, Ptc, and BMP-4 antibodies have been well documented [24–26]. The specificity of the Shh antibody was additionally confirmed by competition with the antigenic peptide at the following concentrations of Shh peptide/Shh antibody per slide: 2/0 µg, 1.5/0.5 µg, 1/1 µg, 0.5/1.5 µg, and 0/2 µg. Sections were stained with diaminobenzidine (DAB) or DAB with nickel and mounted using Crystal Mount (Biomedia, Foster City, CA).

In Situ Hybridization

In situ hybridization was performed as previously described [8, 27] on control (n = 5) and diabetic (n = 5) rat penes that were fixed in 4% paraformaldehyde overnight. A mouse Shh RNA probe was obtained from Andrew McMahon [28], a mouse Ptc RNA probe was obtained from Matthew Scott [29], and a mouse Bmp-4 probe was obtained from Brigid Hogan [30].

Western Analysis

Western analysis was performed on diabetic (n = 5) and control (n = 5) intact rat penes (CD31, {alpha}-actin, Shh, and ß-actin) and isolated corpora cavernosa (Shh) as outlined previously [8]. Penes were homogenized in PBS with the protease inhibitors PMSF, EDTA, and leupeptin, diluted in 2x sample buffer (10 mM Tris, 4% SDS, 200 mM dithiothreitol, 10% glycerol, and 0.2% bromophenol blue) and heated at 95°C for 5 min. Samples were cooled, and protein content was measured by the Lowry method. Proteins were separated via electrophoresis using a 10% polyacrylamide gel and transferred to a 0.45-µ nitrocellulose membrane using a Hoefer Semi-phor Semi-Dry Electroblotter (Amersham Pharmacia, Piscataway, NJ) for 3 h. Membranes were blocked overnight at 4°C in 5% powdered milk in PBS with 0.01% sodium azide. Membranes were incubated with either 250 µl/ml of CD31 (Santa Cruz), {alpha}-actin (Sigma), Shh (Santa Cruz; catalog no. sc-1194), or ß-actin (Sigma) antibodies for 18 h at 4°C. Membranes were washed with PBS-Tween three times for 15 min each time and then incubated with Affini Pure goat anti-mouse IgG conjugated to horseradish peroxidase (1:20 000 dilution; Jackson Immuno Research, West Grove, PA) for 4 h at room temparature. After washing with PBS-Tween, protein bands were visualized using TMB peroxidase substrate (KPL, Gaithersburg, MD) according to the manufacturer's directions and were quantified by densitometry using Kodak 1D software (Rochester, NY). Quantification of bands was performed by comparing the densities of Shh, CD31, and {alpha}-actin to that of ß-actin to eliminate differences in protein loading. Significant differences in protein abundance of Shh/ß-actin, CD31/ß-actin, and {alpha}-actin/ß-actin were determined in control and diabetic penes using a t-test.

Apoptosis

Apoptosis was performed on control (n = 6) and diabetic (n = 6) penes according to the ApopTag kit (Intergen, Purchase, NY). Sections were counted by visual observation of the field with a light microscope. The total number of apoptotic cells in 10 random regions for each penis section was counted. Six sections were counted per animal. Apoptosis was reported as the total number of positively stained cells per visual field. A t-test was performed to determine significant differences in apoptosis between control and diabetic penes.

Bromodeoxyuridine

DNA synthesis was examined in control (n = 3) and diabetic (n = 3) penes by bromodeoxyuridine (BrdU) staining using the BrdU Detection and Labeling Kit II (Roche, Indianapolis, IN). Undiluted BrDU (2 ml/100 g body weight) included in the kit (10 µmoles/L) was injected i.p. into three rats 1 h before they were killed. Penes were excised and fixed in 4% paraformaldehyde overnight at 4°C, processed, and embedded in paraffin. Sections were cut at 8 µm thickness, and BrdU staining was performed as outlined in the Roche kit. Staining was visualized using nitroblue tetrazolium/X-phosphate and light microscopy. Differences in BrdU staining between control and diabetic rat tissues were evaluated visually by an observer blind to the tissue group.

Intracavernosal Pressure Measurements

The intracavernosal pressure (ICP) was measured in control (n = 7) and diabetic (n = 12) rat penes after stimulation, as previously described [31]. Nerves were stimulated (intensity of 6 volts) by placing them on bipolar platinum stimulating electrodes connected to an electrical stimulator (Grass Instruments, Quincy, MA) delivering a series of square wave pulses (1 msec duration at 30 Hz). The cavernous nerve was unilaterally stimulated at a distance of 3 and 5 mm from the major pelvic ganglion. Stimulation lasted 40 sec. A resting interval of at least 5 min separated two consecutive stimulation procedures. The ICP was measured by inserting a 23-ga needle into the corpora cavernosa. A catheter was inserted into the carotid artery for measurement of arterial pressure. These instruments were connected to a pressure transducer. The data were reported as the peak ICP:blood pressure ratio.

Electron Microscopy

Electron microscopy was performed as described previously [32]. Control and diabetic penes were fixed in 2.5% glutaraldehyde, postfixed in 1% OsO4, dehydrated, and embedded in Epon resin. Thin sections were cut and stained with 2% uranyl acetate and 3% lead citrate. Electron microscopy was performed using a JEOL 100CX transmission electron microscope to identify in which cell type apoptosis was taking place.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RT-PCR Analysis of Shh, Ptc, BMP-4, and Hoxd-13 Expression in Control and Diabetic Penes and Pelvic Ganglia

Changes in RNA expression of Shh, Ptc, BMP-4, and Hoxd-13 were quantified by RT-PCR in control and diabetic rat penes (Fig. 2A) and pelvic ganglia (Fig. 2B). Expression of Shh, Ptc, and Hoxd-13 were significantly increased in the diabetic penis (P = 0.00048, 0.027, and 0.01, respectively), but BMP-4 remained unchanged (P = 0.263). Shh, Ptc, and Hoxd-13 expression was also significantly increased in the diabetic pelvic ganglia (P = 0.019, 0.015, and 0.019, respectively), but BMP-4 expression was significantly decreased (P = 0.01).



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  		FIG. 2. The ratios of Shh, Ptc, and Hoxd-13 to GAPDH and of BMP-4 to RPL-32 were evaluated in the rat penis and pelvic ganglia by semiquantitative RT-PCR. A) Shh, Ptc, and Hoxd-13 expression was significantly increased (P = 0.0005, 0.027, and 0.01, respectively) in the diabetic penis, but BMP-4 expression remained unchanged (P = 0.263). B) Shh, Ptc, and Hoxd-13 expression was significantly increased (P = 0.019, 0.015, and 0.019, respectively) in the diabetic pelvic ganglia, but BMP-4 expression was significantly decreased (P = 0.01). Asterisks indicate significant differences in expression

IHC Analysis of Shh, Ptc, and BMP-4 Protein Distribution in Control and Diabetic Penes

The localization of Shh, Ptc, and BMP-4 proteins was examined by IHC in control and diabetic rat penes. Shh was present in the nerves of the dorsal nerve bundle, the epithelium of the urethra [20], and the smooth muscle of penile corpora cavernosal sinusoids (Fig. 3). Shh protein was not observed in the smooth muscle of diabetic penes (Fig. 3). Its localization remained unchanged in the neural and urethral tissues [20]. The specificity of the Shh antibody was confirmed with the antigenic peptide and a no-primary-antibody control (Fig. 4). Ptc protein was present in the epithelium of the urethra [20] and in the smooth muscle of the corpora cavernosal sinusoids (Fig. 3). Ptc protein was absent in the corpora cavernosal sinusoids of the diabetic penes (Fig. 3) but remained abundant in the urethral tissues [20]. BMP-4 protein was localized in the endothelial lining of the corpora cavernosal sinusoids of both control and diabetic penes (Fig. 3).



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  		FIG. 3. IHC analysis of Shh, Ptc, and BMP-4 proteins in control and diabetic rat corpora cavernosa. Shh and Ptc were localized in the smooth muscle of control penile corpora cavernosa. Both Shh and Ptc proteins were not observed in the corpora cavernosa of diabetic penes. BMP-4 protein was identifiable in the endothelial lining of the corpora cavernosal sinusoids of the control penes. BMP-4 protein localization was unaltered in the diabetic penes. x400



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  		FIG. 4. IHC analysis assaying the specificity of the Shh antibody in control rat corpora cavernosa. Shh protein was localized in the smooth muscle of the corpora cavernosal sinusoids. In the absence of primary antibody, staining was not observed (negative control). In the presence of the antigenic peptide (1 µg Shh peptide/1 µg Shh antibody), staining was not identified. Thus, the Shh antibody is specific for Shh protein. x400

In Situ Hybridization of Shh, Ptc, and BMP-4 in Control and Diabetic Penes

The RNA localization of Shh, Ptc, and BMP-4 was examined by in situ hybridization in control and diabetic rat penes. Shh and Ptc expression was abundant in the smooth muscle of the corpora cavernosal sinusoids of both control and diabetic penes (Fig. 5). BMP-4 expression was localized in the endothelial lining of the corpora cavernosal sinusoids of control and diabetic penes (Fig. 5). A change in RNA localization of Shh, Ptc, and BMP-4 was not observed in the diabetic corpora cavernosa.



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  		FIG. 5. In situ hybridization of Shh, Ptc, and BMP-4 in control and diabetic rat corpora cavernosa. Shh and Ptc RNA was localized in the smooth muscle, and BMP-4 RNA was localized in the endothelial lining of the corpora cavernosal sinusoids. RNA localization of Shh, Ptc, and BMP-4 was unaltered in the diabetic penis. x400

IHC Analysis of Smooth Muscle and Endothelial Changes in the Diabetic Penis

IHC analysis was performed on control and diabetic rat penes to examine the effect of diabetes on morphology of the corpora cavernosa. CD31 and {alpha}-actin were used as markers of endothelium and smooth muscle. CD31 staining was abundant in the endothelial lining of the corpora cavernosal sinusoids of control penes (Fig. 6). CD31 staining was not observed in the diabetic corpora cavernosa. The {alpha}-actin distribution remained unchanged in the diabetic penes (Fig. 6).



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  		FIG. 6. IHC analysis of smooth muscle ({alpha}-actin) and endothelial (CD31) markers in control (A and C) and diabetic (B and D) rat corpora cavernosal sinusoids. {alpha}-Actin protein was observed in the smooth muscle of control and diabetic rat penes (A and B, x200). No change in smooth muscle localization was identifiable. Endothelial staining for CD31 was present in control corpora cavernosal sinusoids (C, x100) but was completely absent in diabetic penes (D, x100). Arrows indicate protein localization or where protein would be visible if it were present

Examination of Apoptosis and DNA Synthesis in Control and Diabetic Penes

The amount of apoptosis taking place in control and diabetic penes was measured by TUNEL assay and quantified to evaluate morphological changes in diabetic penes. A significant increase in apoptosis was observed from 32 ± 18 apoptotic cells in control penes (Fig. 7A) to 373 ± 102 apoptotic cells in diabetic penes (Fig. 7B; P = 0.009). The increased apoptosis was accompanied by increased DNA synthesis, as measured by BrdU staining (Fig. 7, C and D).



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  		FIG. 7. Localization of apoptosis and DNA synthesis in control (A and C) and diabetic (B and D) rat penes assayed by TUNEL analysis and BrdU staining. Apoptosis was significantly increased in the diabetic corpora cavernosa (B). A similar increase in BrdU staining was observed in the diabetic corpora cavernosa (D). Arrows indicate apoptosis and BrdU staining. x100

Localization of Apoptosis by TUNEL Assay and Electron Microscopy

Morphological changes of the diabetic penis were evaluated by TUNEL assay. Apoptosis was observed in both the smooth muscle and endothelium of the corpora cavernosa (Fig. 8, A and B). Electron microscopy was performed to confirm which cell type was undergoing apoptosis. Apoptosis was confirmed by electron microscopy in the smooth muscle cells of the corpora cavernosal sinusoids of diabetic penes (Fig. 8). Apoptosis was identified by the presence of chromatin condensation, nuclear fragmentation, and cytoplasmic blebbing, which are common in cells under going apoptosis [33]



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  		FIG. 8. Localization of apoptosis in control (C) and diabetic (A, B, and D) rat penes assessed by TUNEL analysis and electron microscopy. Apoptosis was observed in both the smooth muscle (A, x200) and endothelium lining (B, x400) of the corpora cavernosal sinusoids by TUNEL assay. Arrows indicate apoptotic cells. Comparison of control (C, x6000) and diabetic (D, x6000) smooth muscle by electron microscopy reveals abundant apoptosis in the diabetic smooth muscle. E, Endothelium; SM, smooth muscle. White arrows indicate apoptotic nuclei

Western Analysis of CD31, {alpha}-Actin, and Shh Proteins in Control and Diabetic Penes

Quantification of endothelium and smooth muscle was performed in control and diabetic rat penes by Western analysis to quantify changes in protein abundance that might accompany morphological alterations in the diabetic rat. Both CD31 (endothelium) and {alpha}-actin (smooth muscle) proteins were significantly decreased 4-fold and 1.3-fold, respectively, in diabetic penes (P = 0.001 and 0.026; Fig. 9). Quantification of Shh protein was performed both in isolated corpora cavernosa and in intact penes so that corpora cavernosal changes in Shh protein would not be affected by Shh protein in the urethra, which could obscure small changes in protein abundance in the corpora cavernosa. Shh protein was significantly decreased 1.4-fold in the diabetic corpora cavernosa (P = 0.029; Fig. 9) but was unchanged in the intact penis (control = 1.24 ± 0.21, diabetic = 1.29 ± 0.28; P = 0.35). The specificity of the Shh protein was demonstrated by Western analysis (Fig. 10). These results show that changes in Shh protein were restricted to the corpora cavernosa.



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  		FIG. 9. Western analysis was performed on control (n = 5) and diabetic (n = 5) rat penes assaying for endothelium (CD31, A) and smooth muscle ({alpha}-actin, B). CD31 and {alpha}-actin proteins were both significantly decreased in diabetic penes (P = 0.001 and 0.026 respectively). C) Western analysis was performed on control and diabetic isolated corpora cavernosa assaying for Shh protein. Shh protein was significantly decreased in the diabetic corpora cavernosa (P = 0.029). Protein bands were quantified by densitometry performed by comparing the density of CD31, {alpha}-actin, and Shh bands to that of a ß-actin band to eliminate differences in protein loading. Asterisks indicate significant differences in protein abundance



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  		FIG. 10. Western analysis of Shh protein abundance in isolated penile corpora cavernosa from control (n = 5) and diabetic (n = 5) rat penes. Quantification of Shh protein was performed by comparing the ratio of Shh:ß-actin protein abundance for each lane. Shh protein was significantly decreased in diabetic penile corpora cavernosa (P = 0.029)

ICP Measurement in Control and Diabetic Penes

The ICP was measured in control and diabetic rat penes to evaluate erectile function (Fig. 11). The peak ICP:blood pressure ratio was significantly decreased from an average of 0.91 ± 0.11 in control penes to 0.57 ± 0.08 in diabetic penes (P = 0.009).



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  		FIG. 11. ICP measurement in control and diabetic rat penes after electrical stimulation of the cavernous nerve (stimulation duration was 40 sec). The ICP:blood pressure ratio was dramatically decreased in diabetic penes (P = 0.009)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Diabetic polyneuropathy is a common symptomatic chronic complication of diabetes [34–36]. Peripheral neuropathy affects erectile function by interrupting neural stimulation from the spinal cord to the penis. Compromised penile neural integrity causes abnormal corpora cavernosal morphology [7] and ED [4]. The abnormal morphology includes smooth muscle and endothelial degradation [7] and increased apoptosis [37]. We observed morphological changes in the corpora cavernosa of the BB/WOR diabetic rat penis that are strikingly similar to previous observations of corpora cavernosa morphology after nerve interruption (increased Shh RNA expression in the intact penis, including the smooth muscle of the corpora cavernosa, nerves, and urethra, and decreased Shh protein in the smooth muscle of the corpora cavernosa). A significant decrease in smooth muscle (measured by Western analysis, TUNEL assay, and electron microscopy) and endothelium (measured by Western analysis) of the corpora cavernosal sinusoids was observed. This decrease accompanied a dramatic increase in both apoptosis and DNA synthesis (as indicated by BrdU incorporation), which suggests tissue remodeling. The severe ED (92%) and significant decrease in smooth muscle and endothelium in the BB/WOR rat show that remodeling resulted in a dysfunctional system. The observed anatomical changes in the penile corpora cavernosa were made in an animal model that exhibits neuropathy alone without vasopathy (atherosclerosis and microangiopathy), thus demonstrating that diabetic impotence and the accompanying abnormal corpora cavernosal morphology are correlated with neuropathy alone. Similar smooth muscle and endothelial degradation was observed in the streptozotocin-induced rat model of diabetes, suggesting that neuropathy also is a significant component of that model of insulin-dependent diabetes.

ED is prevalent in the BB/WOR rat model of diabetes. We observed extensive morphological changes in the corpora cavernosa that may account for the observed dysfunction. To understand how penile morphology becomes so disrupted with diabetes that ED ensues, we examined alterations in signaling of a pathway that regulates both homeostasis of the adult penis and differentiation of the corpora cavernosa during postnatal development. A principal component of this regulatory cascade is Shh. Shh functions by regulating cellular proliferation and differentiation [38]. It acts either directly or through induction of secondary signaling molecules (Hox and BMPs) [39, 40]. Targets of Shh signaling that define positional identity are the Hox genes. Other Shh targets are BMP-4 and Ptc. BMP-4 plays a role in interdigital and interductal space formation [41, 42] and has recently been implicated as a regulator of Shh expression [43]. Ptc is the transmembrane receptor for Shh [44]. It is involved in transducing the hedgehog signal and is also a transcriptional target of Shh [45]. Shh, Hoxd-13, BMP-4, and Ptc form part of a cascade of genes that regulate mesenchymal/epithelial interactions during postnatal differentiation and in the adult penis. Disruption of Shh signaling causes abnormal corpora cavernosal morphology, including sinusoid degeneration and smooth muscle and endothelial degradation [20]. These abnormalities can be severe enough to result in ED [20]. Disrupted Shh signaling caused abnormalities similar to those observed in the BB/WOR diabetic rat penis. Thus, interruption of Shh signaling may contribute to the abnormal corpora cavernosal morphology and observed ED present in the diabetic penis.

Shh signaling was significantly altered in the diabetic penis. Shh RNA expression increased in the intact penis, but Shh protein was significantly decreased, as determined by Western analysis, in the isolated corpora cavernosa. The loss of Shh protein was specific to the corpora cavernosa; Shh protein was undetectable in the smooth muscle of the diabetic sinusoids (IHC analysis) but remained unchanged in the intact penis (Western analysis). Unaltered Shh protein abundance in the intact penis showed that Shh protein remained abundant in other areas of the diabetic penis where Shh is expressed, the urethra and the nerves. The distribution of Shh protein in these tissues also remained unchanged in the diabetic rat. Alterations in Shh protein abundance in nerves of the diabetic penis could not be evaluated because of the minute size of the tissue. Because the BB/WOR model of diabetic impotence exhibits peripheral neuropathy, these results suggest that Shh protein in the nerves and in the corpora cavernosa are unrelated and are regulated independently or by different mechanisms. Several possibilities arise as to how Shh signaling becomes altered in the corpora cavernosa without appearing to affect the pool of Shh protein in the neural tissue. The presence of intact neural innervation itself may be required to maintain Shh expression in the corpora cavernosa, or the activity of the nerves or a neurotransmitter derived from the nerves may be required to initiate and maintain normal Shh signaling in the corpora cavernosa indepedent of Shh in the nerve bundle. In previous studies, when the cavernous nerve was cut, Shh protein disappeared from the corpora cavernosa [20]. These findings indicate that neural innervation or some factor from the nerves is required to initiate/maintain the presence of Shh protein in the penile corpora cavernosa.

Increased Shh RNA expression in the penis while Shh protein decreases indicates a block in the process of forming Shh protein from Shh RNA. This block can occur at several steps in the initial synthesis of the 47- to 49-kDa precursor protein (depending on species), during the posttranslational cleavage to yield two mature smaller proteins (19 kDa and 29–31 kDa), or in the attachment of cholesterol to the 19-kDa signaling fragment. The absence of Shh protein in the corpora cavernosal sinusoids may also reflect a change in protein turnover kinetics such that the Shh precursor protein and/or 19-kDa fragment become degraded more quickly. This change in kinetics is possible because the Shh antibody used recognizes the amino terminus of both the precursor and active forms. Ptc, an indicator of Shh function, displays changes similar to those in Shh in the diabetic penis. Hoxd-13, a target of Shh signaling, shows an increase in RNA expression similar to that observed for Shh. Expression of BMP-4, another target of Shh signaling, was significantly decreased in the diabetic pelvic ganglia but not in the penis. The pelvic ganglia are clusters of nerve cell bodies that are remote from the penis (near the prostate). They provide innervation to the penis via the cavernous nerve (axon that provides innervation from the pelvic ganglia to the dorsal nerve bundle of the penis). The differences in BMP-4 RNA expression between the pelvic ganglia and the corpora cavernosa occur because these areas represent separate pools of BMP-4 expression. Ribosomes are not present in the cavernous nerve, so only the BMP-4 protein made in the pelvic ganglia may travel down the nerve. Because BMP-4 protein was not observed in the nerves of the dorsal nerve bundle under any conditions, the BMP-4 RNA and protein observed in the corpora cavernosa probably were synthesized directly in this tissue and did not derive from the pelvic ganglia. This is not the case with Shh; Shh protein was abundant in the nerves of the dorsal nerve bundle, thus indicating some interaction between the pelvic ganglia and corpora cavernosal smooth muscle. BMP-4 is important during development because of its role in apoptosis [42] and was included in this study because Shh is a regulator of proliferation through induction of secondary signaling molecules such as Hox and BMPs, which have a positive and negative effect on proliferation, respectively. Because BMP-4 is important in the process of apoptosis, its regulation is likely multifactoral. BMP-4 protein is localized in the endothelial lining of the corpora cavernosal sinusoids of both control and diabetic penes. Although Western analysis of CD31 indicates that endothelial tissue is decreased or dysfunctional (not expressing normal endothelial markers) in the diabetic penis, significant DNA synthesis (indicated by BrdU staining) is also occurring as the penis attempts to maintain structural integrity. BMP-4 is likely involved in the process of tissue regrowth (tissue specification) that is taking place in the sinusoids to reestablish normal morphology, and thus its expression may be maintained or elevated in the regenerating tissue and thus compensate for decreased or dysfunction endothelium. Thus, BMP-4 RNA levels remain unchanged in the intact penis. BMP-4 protein levels also may decrease in the penis as endothelial degradation occurs; however, BMP-4 protein abundance was not evaluated in this study. These findings suggest that in addition to the affect on regulation of Shh in the diabetic penis, there is probably a disruption of an entire signaling cascade involving Ptc, Hox, and BMP-4, which maintains normal corpora cavernosal morphology in the adult.

In a previous study, we examined the role of abnormal Shh function in ED and Shh signaling after cavernous nerve injury [20]. We injected Affi-gel beads soaked in an inhibitor of Shh function (5E1 Shh inhibitor; Jessel, Hybridoma Bank, University of Iowa, Ames, IA) directly into the corpora cavernosa of the penis. After 7 days, the corpora cavernosal tissue appeared dedifferentiated in the treated region; sinusoid structure was completely absent. The morphology of the corpora cavernosa was dramatically affected, so much so that erectile function measured by ICP was severely decreased [20]. Thus, inhibition of Shh function is correlated with ED. There are several sources of Shh protein in the penis, including the nerves of the dorsal nerve bundle, the smooth muscle of the corpora cavernosa, and the urethra [20]. One method of inhibiting Shh function in vivo is to surgically cut the cavernous nerve, which innervates the penis. Shh RNA expression increased and Shh protein decreased in the intact penis (smooth muscle of the corpora cavernosa, nerves, and urethra) with nerve injury. Morphological changes in the corpora cavernosal smooth muscle, one site of Shh distribution, and ED were prevalent in this model. Thus, a correlation of Shh function, neural integrity, and corpora cavernosal homeostasis necessary for erection was identified. In the present study, we examined another model of neuropathy that also results in profound ED, the diabetic BB/WOR rat. In this model, Shh RNA expression increased and Shh protein disappeared in the diabetic penis. We saw strikingly similar changes in both models of neuropathy and ED. Whether Shh dysfunction is a symptom of neuropathy or is causal has yet to be determined. It is clear that Shh signaling becomes altered with neuropathy and that alteration of Shh function can lead to ED because of morphological changes in the corpora cavernosa. How Shh signaling is regulated in the penis and how Shh regulation becomes altered in the diabetic rat and more specifically, with neuropathy, are currently under investigation. Increasing our understanding of the molecular mechanisms that regulate Shh signaling may provide valuable insight into improving treatment options for patients with diabetic impotence.


   ACKNOWLEDGMENTS
 
The authors thank Andrew McMahon, Matthew Scott, and Brigid Hogan for supplying Shh, Ptc, and BMP-4 constructs to synthesize riboprobes and Alfred Rademaker for assistance with statistical analysis.


   FOOTNOTES
 
1 Supported by the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases, grants DK54478, DK55046, DK59071, and DK62970. Back

2 Correspondence: Carol Podlasek, Department of Urology, Northwestern University, Tarry Building 11-715, 303 E. Chicago Ave., Chicago, IL 60611. FAX: 312 908 7275; cap325{at}northwestern.edu Back

Received: 15 November 2002.

First decision: 16 December 2002.

Accepted: 30 April 2003.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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