Constitutive LH receptor activity impairs NO-mediated penile smooth muscle relaxation

in Reproduction
Authors:
Deepak S HiremathDepartment of Physiology, Southern Illinois School of Medicine, Carbondale, Illinois, USA

Search for other papers by Deepak S Hiremath in
Current site
Google Scholar
PubMed
Close
,
Fernanda B M PrivieroCardiovascular Translational Research Center and Department of Cell Biology and Anatomy University of South Carolina, Columbia, South Carolina, USA

Search for other papers by Fernanda B M Priviero in
Current site
Google Scholar
PubMed
Close
,
R Clinton WebbCardiovascular Translational Research Center and Department of Cell Biology and Anatomy University of South Carolina, Columbia, South Carolina, USA

Search for other papers by R Clinton Webb in
Current site
Google Scholar
PubMed
Close
,
CheMyong KoDepartment of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

Search for other papers by CheMyong Ko in
Current site
Google Scholar
PubMed
Close
, and
Prema NarayanDepartment of Physiology, Southern Illinois School of Medicine, Carbondale, Illinois, USA

Search for other papers by Prema Narayan in
Current site
Google Scholar
PubMed
Close
https://orcid.org/0000-0002-6468-5964

Correspondence should be addressed to P Narayan; Email: pnarayan@siu.edu
Free access

Timely activation of the luteinizing hormone receptor (LHCGR) is critical for fertility. Activating mutations in LHCGR cause familial male-limited precocious puberty (FMPP) due to premature synthesis of testosterone. A mouse model of FMPP (KiLHRD582G), expressing a constitutively activating mutation in LHCGR, was previously developed in our laboratory. KiLHRD582G mice became progressively infertile due to sexual dysfunction and exhibited smooth muscle loss and chondrocyte accumulation in the penis. In this study, we tested the hypothesis that KiLHRD582G mice had erectile dysfunction due to impaired smooth muscle function. Apomorphine-induced erection studies determined that KiLHRD582G mice had erectile dysfunction. Penile smooth muscle and endothelial function were assessed using penile cavernosal strips. Penile endothelial cell content was not changed in KiLHRD582G mice. The maximal relaxation response to acetylcholine and the nitric oxide donor, sodium nitroprusside, was significantly reduced in KiLHRD582G mice indicating an impairment in the nitric oxide (NO)-mediated signaling. Cyclic GMP (cGMP) levels were significantly reduced in KiLHRD582G mice in response to acetylcholine, sodium nitroprusside and the soluble guanylate cyclase stimulator, BAY 41-2272. Expression of NOS1, NOS3 and PKRG1 were unchanged. The Rho-kinase signaling pathway for smooth muscle contraction was not altered. Together, these data indicate that KiLHRD582G mice have erectile dysfunction due to impaired NO-mediated activation of soluble guanylate cyclase resulting in decreased levels of cGMP and penile smooth muscle relaxation. These studies in the KiLHRD582G mice demonstrate that activating mutations in the mouse LHCGR cause erectile dysfunction due to impairment of the NO-mediated signaling pathway in the penile smooth muscle.

Abstract

Timely activation of the luteinizing hormone receptor (LHCGR) is critical for fertility. Activating mutations in LHCGR cause familial male-limited precocious puberty (FMPP) due to premature synthesis of testosterone. A mouse model of FMPP (KiLHRD582G), expressing a constitutively activating mutation in LHCGR, was previously developed in our laboratory. KiLHRD582G mice became progressively infertile due to sexual dysfunction and exhibited smooth muscle loss and chondrocyte accumulation in the penis. In this study, we tested the hypothesis that KiLHRD582G mice had erectile dysfunction due to impaired smooth muscle function. Apomorphine-induced erection studies determined that KiLHRD582G mice had erectile dysfunction. Penile smooth muscle and endothelial function were assessed using penile cavernosal strips. Penile endothelial cell content was not changed in KiLHRD582G mice. The maximal relaxation response to acetylcholine and the nitric oxide donor, sodium nitroprusside, was significantly reduced in KiLHRD582G mice indicating an impairment in the nitric oxide (NO)-mediated signaling. Cyclic GMP (cGMP) levels were significantly reduced in KiLHRD582G mice in response to acetylcholine, sodium nitroprusside and the soluble guanylate cyclase stimulator, BAY 41-2272. Expression of NOS1, NOS3 and PKRG1 were unchanged. The Rho-kinase signaling pathway for smooth muscle contraction was not altered. Together, these data indicate that KiLHRD582G mice have erectile dysfunction due to impaired NO-mediated activation of soluble guanylate cyclase resulting in decreased levels of cGMP and penile smooth muscle relaxation. These studies in the KiLHRD582G mice demonstrate that activating mutations in the mouse LHCGR cause erectile dysfunction due to impairment of the NO-mediated signaling pathway in the penile smooth muscle.

Introduction

The luteinizing hormone receptor (LHCGR), a member of G-protein coupled receptor family, has a critical role in steroidogenesis and spermatogenesis in males (Ascoli et al. 2002, Narayan et al. 2019). Constitutively activating mutations in LHCGR resulting in single amino acid replacements and inherited in an autosomal dominant male-limited pattern causes a condition called familial male-limited precocious puberty (FMPP). The disorder is characterized by elevated testosterone levels, Leydig cell hyperplasia and precocious puberty in boys by 3–4 years of age (Themmen & Huhtaniemi 2000, Ulloa-Aguirre et al. 2014). The most common mutation identified in FMPP is the mutation of aspartic acid at amino acid residue 578 to glycine (D578G). To understand the role of constitutively activated LHCGR in reproductive function, we previously developed a knock-in mouse model (KiLHRD582G) by introducing an aspartic acid to glycine mutation at amino acid residue 582 (D582G) in mouse LHCGR, corresponding to the D578G in human LHCGR. KiLHRD582G mice exhibit similar phenotypes seen in FMPP patients such as precocious puberty, supraphysiological levels of testosterone and Leydig cell hyperplasia (McGee & Narayan 2013).

Our previous studies showed that KiLHRD582G mice became progressively infertile by 6 months of age in spite of normal sperm count and motility (McGee & Narayan 2013, Hai et al. 2017). Mating studies with superovulated females demonstrated that KiLHRD582G mice had viable sperm and normal accessory gland function. Sexual behavior studies revealed that KiLHRD582G male mice had normal mounting behavior but exhibited a longer latency to first intromission and a significantly shorter duration of intromission with an inability to ejaculate, suggesting erectile dysfunction (ED) (Hai et al. 2017). Morphological changes observed in the penile corpora cavernosa of KiLHRD582G mice included reduced smooth muscle content and chondrocyte accumulation without a change in collagen content (Hai et al. 2017). These changes are mediated by high levels of testosterone (Hiremath et al. 2020). However, the functional consequences of the morphological changes in the penis of the KiLHRD582G mice were not determined.

The penile corpora cavernosal smooth muscle lining the sinusoids plays a key role in penile erection (Dean & Lue 2005, Traish 2009). Various studies have demonstrated that a significant decrease in cavernosal smooth muscle causes impaired erectile response (Nehra et al. 1998, Ahn et al. 2005, Mostafa et al. 2013, Lombo et al. 2016). During sexual stimulation, the release of nitric oxide (NO) from the non-adrenergic/cholinergic nerves causes dilation of the cavernosal arteries and relaxation of the sinusoidal smooth muscle causing increased blood flow into the penis and resulting in further production of NO from endothelial cells. Expansion of the sinusoids and the connective tissue matrix causes compression of the subtunical venules and reduces blood outflow (veno-occlusion) resulting in increased intracavernosal pressure and erection (Dean & Lue 2005, Nunes & Webb 2012). Thus, smooth muscle relaxation as well as the architecture of the extracellular matrix are important for erectile function.

The major pathway for smooth muscle relaxation is the NO-cyclic GMP (cGMP) pathway (Dean & Lue 2005, Morelli et al. 2006, Nunes & Webb 2012). NO released from the cavernous nerves and endothelial cells activates soluble guanylate cyclase (sGC) in the smooth muscle cells, increasing cGMP which activates protein kinase G (PRKG1). Activated PRKG1 decreases intracellular levels of calcium causing smooth muscle relaxation (Dean & Lue 2005, Andersson 2011). Noradrenaline from sympathetic nerves and endothelin and prostaglandin F2α from endothelial cells increase intracellular levels of calcium causing smooth muscle contraction (Traish et al. 2000, Dean & Lue 2005, Morelli et al. 2006). In addition, the RhoA/Rho-associated protein kinase (ROCK) pathway maintains contractile tone (Sopko et al. 2014). Dysregulation of these pathways has been shown to contribute to ED (Akingba & Burnett 2001, Bivalacqua et al. 2004, Chiou et al. 2010, Toque et al. 2013).

We hypothesize that premature and chronic activation of LHCGR in the KiLHRD582G mouse causes impaired smooth relaxation resulting in erectile dysfunction. To test this hypothesis, animal behavioral studies and investigation of smooth muscle relaxation and contraction pathways were performed. Our results show that infertility caused by constitutive activation of LHCGR is due to erectile dysfunction as a result of reduced NO-mediated production of cGMP resulting in decreased penile smooth muscle relaxation.

Materials and methods

Animal care

The generation of the KiLHRD582G mouse model was described previously (McGee & Narayan 2013). KiLHRD582G mice (B6129S-Lhcgrtm1.1Pnara/J) are available from the Jackson Laboratory as JAX#029311. KiLHRD582G male mice were bred with B6129SF1/J hybrid female mice (Jackson Laboratory) to obtain heterozygous KiLHRD582G male mice and WT littermates. Mice were maintained on 12 h light:12 h darkness cycle and fed a standard chow diet (Purina LabDiet 5008) and tap water. Male mice between 26 and 36 weeks of age were used for the studies. Mice were euthanized by CO2 asphyxiation, followed by decapitation. Breeding of animals was done according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All animal studies were approved by the Institutional Animal Care and Use Committee at Southern Illinois University.

Apomorphine-induced erection studies

Each mouse was placed in a plexiglass chamber alone for a period of 30 min to acclimatize to the new chamber. A single subcutaneous injection of saline was then administered, and the mouse was observed for 30 min by video recording. An injection of R-(−)-apomorphine hydrochloride hemihydrate (A4393, Sigma Aldrich) at an optimal dose of 3.2 µg/kg dissolved in 0.1% ascorbic acid was administered subcutaneously to the same mouse and the mouse was recorded again for a period of 30 min. Penile erections were identified in these recordings by the criteria described previously (Rampin et al. 2003). An erection was scored when the mouse stood up on its hindlimbs, bent its head toward the penis, held the penis and licked it with hip movements.

Real-time quantitative PCR

The penile body was separated from the glans penis and the cavernosa was isolated after removal of the urethra and nerve bundle under a dissecting microscope and snap-frozen in liquid nitrogen. The cavernosal tissue samples were homogenized in TRIzol (Invitrogen) and RNA was extracted according to the manufacturer’s instructions. Synthesis of cDNA was done using M-MLV Reverse Transcriptase (Promega). RT-qPCR was performed using PowerUP SYBR Green Master mix (Applied Biosystems). Primers were designed to span an exon–exon junction to ensure amplification of only the cDNA. The sequences of the primers are shown in Table 1. Samples were analyzed in duplicate and normalized to ribosomal protein S2 (Rps2). A template negative control and a calibrator sample, prepared by mixing equal amounts of cDNA from all the samples, were also analyzed. Data are expressed as relative to the calibrator sample using the 2−ΔΔCT method as previously described (McGee & Narayan 2013).

Table 1

Primers used in quantitative RT-PCR.

Gene Primer sequence RefSeq number
Forward Reverse
Col1a1 GCG GTA ACG ATG GTG CTG TT GCT TCA CCC TTA GCA CCA ACT NM_007742.4
Col1a2 CAT TAT CTG TGA AGA CCC AGA CTGC CTT TCT GCC CCT TTG GCC CTA NM_007743.3
Col3a1 ACA GAG GAG AAA CTG GCC CT ACC TTT GTC ACC TCG TGG AC NM_009930.2
Eln ATC AAA GCA CCA AAG CTG CC CTG CAC CAG CTA CTC CAT AGG NM_007925.4
Fbn1 ATC CTT GTG AGT TGT GCC CC CGT CCA TAT CAA CTG CTG AGTC NM_007993.2
Fbn2 CGG AGC CAA ATC AAT TCA GCA CCC GTT CTG GCA TCC ATT CT NM_010181.2
Nos1 CAC AGA TGA GGT TTT CAG CTC C CGT GTG TGT CCC CGT TTA GT NM_008712.3
Nos3 ATG CTG CTA GAA ATC GGG GG CAC ACA GCC ACA TCC TCA AG NM_008713.4
Pde5a CAC AGG GAA GAG GTT GTT GG ACC ACA GAA TGC CAG GTA GG NM_153422.2
Prkg1 AAG AGC CCA CAG TCG AAG GA CCG TAT TCC ACG GGG TAC AT NM_011160.3
Rhoa CTC CGT CGG TTC TCT CCA TA ATG CAA GGC TCA AGG CGAG NM_016802.5
Rock1 TGT TGG CAA TCA GCT ACC TT CTT TCC TGC AAG CTT TTA TCC A NM_009071.2
Rock2 GTT CAG TTG GTT CGT CAT AAG GC GGC CAT AAT ATC TCT TTC TTC CCAG NM_009072.2
Rps2 CCA GGT TCA AGG CTT TCGT GCA CAG GGA CGA TGG AAA NM_008503

Functional studies in cavernosal strips

Penes from mice were dissected and placed in chilled physiological salt solution (PSS) of the following composition: 130 mM NaCl, 4.7 mM KCl, 1.18 mM KH2PO4, 1.18 mM MgSO4, 14.9 mM NaHCO3, 5.6 mM glucose and 1.56 mM CaCl2. The penile body was separated from the glans penis and the cavernosa was isolated after removal of the urethra and nerve bundle under a dissecting microscope. A slit was made along the shaft of the cavernosa to obtain two strips and one strip from each animal was snap-frozen in liquid nitrogen for Western blot analysis and the other strip was mounted on pins in a myograph chamber (Danish Myograph Technology, Aarhus, Denmark) coupled to a PowerLab data acquisition system (Software chart 5.0, AD Instruments, Colorado Springs, USA). The strips were equilibrated in PSS at 37°C for 30 min bubbled with a mixture of 95% O2 and 5% CO2. Subsequently, a resting tension of 5 mN was applied for 1 h. To assess the contractile ability of the strips, PSS containing 120 mM potassium (KPSS) was added. Cumulative concentration-dependent contraction response was obtained by adding 10−9 to 10−4 M of the α1 adrenergic agonist, phenylephrine (PE, Sigma Aldrich). Concentration-dependent relaxation responses to 10−9 to 10−6 M acetylcholine (ACh, Cayman Chemical), 10−9 to 10−5 M of the NO donor, sodium nitroprusside (SNP, Sigma Aldrich) and 10−9 to 10−4 M of the Rho-kinase inhibitor, Y-27632 (Tocris, Minneapolis, MN) were obtained in tissue strips precontracted with 10−5 M PE. All stock solutions were prepared in deionized water except Y-27632 which was prepared in DMSO.

Western blot analysis

Frozen penile cavernosal strips from WT and KiLHRD582G mice were obtained as described previously. Each strip was then pulverized and sonicated in RIPA lysis buffer (sc-24948, Santa Cruz Biotechnology). Sonication was performed on ice under the following conditions: six short bursts at power 4 W for 10 s and four short bursts at power 4 W for 5 s with a cooling period of 30 s between every burst. Samples were then centrifuged at 13,000 g for 15 min and the supernatant was collected to estimate protein concentration using the Coomassie (Bradford) assay kit (Thermofisher Scientific). Forty micrograms of total protein were resolved on 8% SDS-PAGE and the bands were transferred to Immobilin-FL PVDF membrane (Millipore-Sigma) at 100 V for 1.5 h. The membrane was then blocked with Odyssey blocking buffer diluted 1:1 in Tris-buffered saline (TBS) (LI-COR Biosciences, Lincoln, NE) and probed with either anti-rabbit monoclonal NO synthase3 (NOS3 or eNOS, 1:100, #32027, Cell Signaling Technology), polyclonal cGMP specific phosphodiesterase type 5 (PDE5, 1:100, #2395, Cell Signaling Technology), polyclonal NO synthase 1 (NOS1 or nNOS, 1:100, #4234, Cell Signaling Technology) or monoclonal protein kinase G (PRKG1,1:100, # 3248, Cell Signaling Technology) and anti-goat glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000, AF5718, R&D Systems, Minneapolis, MN) in 5% BSA in TBS/0.1% Tween-20 (TBST) overnight at 4°C. The membrane was washed three times with TBST for 5 min each followed by incubation with donkey anti-rabbit conjugated with Alexa Flour 790 (1:2000) and donkey anti-goat conjugated with Alexa Flour 680 (1:10,000) secondary antibodies (Invitrogen) in 0.01% SDS in TBST for 1 h at room temperature. The membrane was washed three times with TBST for 5 min each, followed by a final rinse in TBS and scanned using the Odyssey CLx infrared imaging system (LI-COR Biosciences, Lincoln, NE). The intensities of the specific bands were quantified using LI-COR Image Studio 3.1 software and normalized to the intensity of the loading control, GAPDH.

Immunohistochemistry

Penile body tissue samples were collected and fixed in modified Carnoy’s solution (60% methanol, 30% ethanol and 10% acetic acid) for 4 hours at room temperature, rinsed briefly in PBS and stored in 70% ethanol at 4°C before processing. Tissue samples were embedded in paraffin and 5 µm sections were prepared. Sections were blocked with 10% normal goat serum for 1 h at room temperature was done followed by incubation with primary rat mouse pan-endothelial cell antigen (MECA-32) antibody overnight at 4°C (1:2 dilution, kindly provided by Dr Sophia Ran, Southern Illinois University, School of Medicine). Sections were washed in TBST for 2 x 5 min and incubated in biotinylated secondary goat anti-rat antibody (1:200, Vector Laboratories, Burlingame, CA). Antigen-antibody complexes were visualized using Vectastain Elite ABC-DAB kit (Vector Laboratories) and counterstained with hematoxylin. Quantification of MECA-32 stained area in the corpora cavernosa was performed on four non-serial sections, each 25 µm apart, from each animal using ImageJ software (NIH).

Determination of cGMP levels

Penile cavernosal tissues were dissected and equilibrated in PSS for 1 h as described above. Tissue strips were precontracted with 10−5 M PE and relaxed with either 10−5 M SNP, 10−6 M ACh, or 10−5 M BAY 41-2272 and snap-frozen immediately in liquid nitrogen after relaxation. For penile cGMP content, tissues were pulverized and homogenized in 5% trichloroacetic acid, centrifuged and extracted in water-saturated ether. cGMP levels were determined by the ELISA kit (Cayman, Chemical, Ann Arbor, MI) according to manufacturer’s instructions and were normalized to the weight of the cavernosal strip.

Statistical analysis

Relaxation responses to drugs were expressed as percentage of submaximal contraction induced by PE. Curves were fitted to data using non-linear regression using Prism 5 software program (Graphpad Software Inc.). Drug maximum responses and potencies were calculated as Emax (maximal response produced by the drug) and pEC50 (negative logarithm of molar concentration of drug that produced 50% of maximal response). Statistical significance of differences between WT and KiLHRD582G mice was determined by unpaired Student’s t-test using Prism 5 software. Outliers were identified using Grubbs test online (http://www.graphpad.com/quickcalcs/Grubbs1.cfm). P < 0.05 was considered statistically significant.

Results

KiLHRD582G mice have erectile dysfunction

In our previous study (Hai et al. 2017), we demonstrated that KiLHRD582G male mice exhibit progressive infertility and sexual behavior studies suggested KiLHRD582G mice might have developed erectile dysfunction. To establish if the KiLHRD582G mice develop erectile dysfunction, an erectile response to the dopamine receptor agonist, apomorphine, was determined. A systemic administration of apomorphine elicits penile erection in a number of species including mice and has been widely used as a way to determine erectile responsiveness (Giuliano & Allard 2001, Giuliano et al. 2002, Matsumoto et al. 2005, Simonsen et al. 2016). Analysis of the video recording indicated that nine out of eleven (82%) WT mice injected with apomorphine exhibited erections, compared to one out of thirteen (8%) KiLHRD582G mice (Table 2). Further, the number of erections per mouse was significantly lower in KiLHRD582G mice compared with WT mice. No spontaneous erections were observed in either WT or KiLHRD582G mice injected with saline during the 30-minute recording period. These results demonstrate that apomorphine was unable to elicit erections in KiLHRD582G mice indicating that KiLHRD582G mice had erectile dysfunction.

Table 2

Effects of apomorphine on penile erection. Data are presented as mean ± s.e.m.

Genotype/treatment No. of responders No. of erections/mouse
WT (n = 11)
 Saline 0/11 0
 Apomorphine 9/11 1.18 ± 0.26
KiLHRD582G (n = 13)
 Saline 0/13 0
 Apomorphine 1/13 0.15 ± 0.15**

**P < 0.01 compared to WT apomorphine treatment.

Expression of key components of the extracellular matrix is not altered in the corpora cavernosa of KiLHRD582G mice

The extracellular matrix (ECM) of the corpora cavernosa is mainly composed of collagen, elastic fibers and proteoglycans. Changes in these components could alter the structural integrity of the corpora cavernosa leading to erectile dysfunction (Sattar et al. 1994, Traish et al. 2003, Costa et al. 2006, Luttrell et al. 2008, Wang et al. 2015). We have previously demonstrated that total collagen content, determined by the hydroxyproline assay, was unchanged in KiLHRD582G mice (Hai et al. 2017). To determine if there were changes in specific components of the extracellular matrix, we measured the mRNA expression levels of the α1 and α2 subunits of collagen I (Col1a1 and Col1a2) and collagen III (Col3a1), the major collagen isoforms in the mouse corpora cavernosa, elastin (Eln), the core component of elastic fibers and fibrillin (Fbn1 and Fbn2), which provide a microfibril scaffold for elastin. Their expression levels in the corpora cavernosa were not significantly different between WT and KiLHRD582G mice (Fig. 1), suggesting that impairment in erectile function in the KiLHRD582G mice was not due to alterations in the extracellular matrix.

Figure 1
Figure 1

Normal collagen, elastin and fibrillin RNA levels in the cavernosa of KiLHRD582G mice. Relative expression of Col1a1, Col1a2, Col3a1, Eln, Fbn1 and Fbn2 represents levels of RNA normalized to internal control Rps2 and relative to calibrator sample prepared by mixing equal amounts of cDNA from all samples. Data are expressed as mean ± s.e.m. (n = 5–6).

Citation: Reproduction 161, 1; 10.1530/REP-20-0447

The corpora cavernosa of KiLHRD582G mice have normal endothelial cell content and function

Endothelial cells in the cavernosa produce NOS3 (eNOS), which plays an essential role in maintaining penile erection by generation of NO. Loss of endothelial cells can contribute to an impairment in penile erection (Burchardt et al. 2000, Liu et al. 2013). To determine if there was a reduction in endothelial cell content in corpora cavernosa of KiLHRD582G mice, penile body sections from 26-week-old WT and KiLHRD582G mice were stained with an antibody against an endothelial cell-specific marker MECA-32 (Fig. 2A). Immunohistochemical quantification by ImageJ analysis did not show a significant change in the stained area for MECA-32 in the cavernosa between WT and KiLHRD582G mice (Fig. 2B). To confirm these findings, quantitative real-time PCR and Western blot analysis were performed to determine the levels of NOS3, another endothelial cell marker. The RNA and protein levels of NOS3 in the cavernosa of KiLHRD582G mice were not significantly different compared to WT mice (Fig. 2C,D and E). To assess endothelial cell function, endothelium-dependent relaxation to ACh was measured. The EC50 value for relaxation was not different between the WT and KiLHRD582G tissues (Fig. 2F and Table 3). However, maximal relaxation (Emax) to ACh was significantly reduced in the KiLHRD582G mice compared with WT mice which could be due to reduced smooth muscle content and/or function. Together, these results indicate normal endothelial cell content and function in the cavernosa of KiLHRD582G mice.

Figure 2
Figure 2

Normal endothelium content in the cavernosa of KiLHRD582G mice. (A) Representative photomicrographs of immunohistochemical staining of MECA-32 in WT (n = 4) and KiLHRD582G (n = 6) mice. Scale bars, 100 µm. (B) Quantification of MECA-32 stained area in the corpora cavernosa of WT and KiLHRD582G mice determined by Image-J analysis. (C) Quantification of Nos3 by RT-qPCR (n = 5). Relative expression represents levels of RNA normalized to internal control Rps2 and relative to calibrator. (D) Western blot image for NOS3 protein levels. GAPDH was used as the loading control. M, Molecular weight markers, lanes 1, 3, 5, 7 – WT and lanes 2, 4, 6, 8 – KiLHRD582G (n = 4). Expected size of NOS3 is 140 kDa. (E) Levels of NOS3 relative to GAPDH was determined by quantification of intensities of bands on Western blots. (F) Concentration-dependent relaxation response induced by ACh (n = 6). Data are expressed as mean ± s.e.m. ***P < 0.001 compared to WT.

Citation: Reproduction 161, 1; 10.1530/REP-20-0447

Table 3

Summary of functional studies in cavernosal strips.

Drug WT KiLHRD582G
Emax pEC50 Emax pEC50
Phenylephrine 0.16 ± 0.01 mN/mg 5.46 ± 0.054 0.06 ± 0.01 mN/mg**** 5.52 ± 0.14
Sodium nitroprusside 88.8 ± 1.9% 6.17 ± 0.052 68.3 ± 3.4%*** 5.95 ± 0.057*
Acetylcholine 69.5 ± 3.6% 6.76 ± 0.088 30.0 ± 5.5%*** 6.75 ± 0.24
Y-27632 84.6 ± 1.4% 6.01 ± 0.072 77.2 ± 1.2%** 5.85 ± 0.055

Values are expressed as mean ± s.e.m. (n = 6).

*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to WT.

Emax, Maximum response elicited by the drug; pEC50, negative logarithm of molar concentration of drug that produced 50% of maximal response.

KiLHRD582G mice have smooth muscle dysfunction in the corpora cavernosa

We next determined if smooth muscle function was altered in the corpora cavernosa of KiLHRD582G mice. For this, concentration-dependent contraction to the α1 adrenergic agonist, PE and relaxation of PE-contracted tissue to the NO donor, SNP were measured in cavernosal strips. The maximal contractile and relaxation responses to PE and SNP, respectively were significantly reduced in the KiLHRD582G mice compared with WT mice (Fig. 3A, B and Table 3), suggesting decreased smooth muscle function consistent with the reduction with smooth muscle content (Hai et al. 2017). The potency of the contractile response to PE was not different between the WT and KiLHRD582G mice. However, the EC50 value for SNP was significantly higher (1.7-fold) in the KiLHRD582G mice (Fig. 3B and Table 3). Together, these data suggest impairment in the NO-mediated relaxation pathway.

Figure 3
Figure 3

Smooth muscle dysfunction in the cavernosal segments of WT and KiLHRD582G mice. (A) Concentration-dependent contractile response induced by PE. (B) Concentration-dependent relaxation response induced by SNP. Data are expressed as mean ± s.e.m. (n = 6). ***P < 0.001, ****P < 0.0001 compared to WT.

Citation: Reproduction 161, 1; 10.1530/REP-20-0447

KiLHRD582G mice have normal levels of NO signaling pathway intermediates

As NO-cGMP-mediated smooth muscle relaxation is the major erectile pathway in the corpora cavernosa, we examined the RNA and protein levels of enzymes that are required for production and degradation of cGMP or activated by cGMP. Neuronal nitric oxide synthase (NOS1) and NOS3 (examined previously in Fig. 2) are required for the production of NO from nonadrenergic–noncholinergic cavernous nerves and endothelial cells, respectively, and NO is required for the production of cGMP. PDE-5 rapidly degrades cGMP. PRKG1 is a key downstream intermediate that is activated by cGMP and in turn phosphorylates various proteins and ion channels to decrease intracellular calcium and cause smooth muscle relaxation (Dean & Lue 2005, Nunes & Webb 2012). Determination of the RNA levels of these intermediates showed that there was no change in RNA levels of Nos1 and Prkg1 but Pde5a was significantly reduced in the cavernosa of KiLHRD582G mice (Fig. 4A,C and E). Protein levels of NOS1 and PRKG1 were not different between the cavernosa of WT and KiLHRD582G mice but PDE5 levels were significantly reduced in the cavernosa of KiLHRD582G mice (Fig. 4B, D and F). PDE5 is the only intermediate examined that is specific to smooth muscle cells in the penis and no change in RNA levels was seen between WT and KiLHRD582G mice when the expression was normalized to smooth muscle actin (data not shown).

Figure 4
Figure 4

Normal RNA and protein levels of signaling intermediates in the NO signaling pathway for smooth muscle relaxation in the cavernosa of KiLHRD582G mice. Quantification of Nos1 (A), Pde5a (C) and Prkg1 (E) by RT-qPCR (n = 5). Representative Western blot images for NOS1 (B), PDE5 (D), PRKG1 (F) protein levels. GAPDH was used as the loading control. Quantification of the level of each protein relative to GAPDH was determined by quantification of intensities of bands on Western blots (n = 4). M, molecular weight markers. Expected sizes of NOS1, PDE5, PRKG1 and GAPDH are 160, 100, 78 and 37 kDa, respectively. Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01 compared to WT.

Citation: Reproduction 161, 1; 10.1530/REP-20-0447

The Rhokinase signaling pathway is not altered in KiLHRD582G mice

To determine if upregulation of Rho-kinase pathway contributed to impairment of smooth muscle relaxation, a concentration-dependent relaxation curve to the Rho-kinase inhibitor, Y-27632 was performed (Fig. 5A). The maximal relaxation response was significantly reduced in KiLHRD582G mice, without a change in the potency (Table 3). Additionally, RNA levels of the Rho-kinase pathway signaling intermediates, Rhoa, Rock1, Rock2, were not altered in KiLHRD582G mice compared with WT mice (Fig. 5B, C and D). Together, these results suggest that the reduced relaxation response in the KiLHRD582G mice is not due to a dysregulated Rho-kinase pathway.

Figure 5
Figure 5

Normal gene expression levels of signaling intermediates of the Rho-kinase smooth muscle contractile pathway in the cavernosa of KiLHRD582G mice. (A) Relaxation response induced by Y-27632, a Rho-kinase inhibitor (n = 6). Quantification of Rhoa (B), Rock1 (C), Rock2 (D) RNA by RT-qPCR (n = 5). Relative expression represents levels of RNA normalized to the internal control Rps2 and relative to the calibrator sample prepared by mixing equal amounts of cDNA from all samples. Data are expressed as mean ± s.e.m. **P < 0.01 compared to WT.

Citation: Reproduction 161, 1; 10.1530/REP-20-0447

KiLHRD582G mice exhibit reduced activation of soluble guanylyl cyclase (sGC)

Since the signaling intermediates of NO pathway were not altered and the maximal relaxation responses to ACh and SNP were lower, we examined the activity of sGC by measuring cGMP levels in response to ACh and SNP. The basal levels of cGMP were not different between the WT and KiLHRD582G mice (Fig. 6). The response to ACh and SNP was reduced in KiLHRD582G mice compared to WT controls, suggesting that NO-mediated activation of sGC was impaired in the KiLHRD582G mice. BAY 41-2272, a NO independent stimulator of sGC, increased cGMP levels in WT and KiLHRD582G mice over that obtained with SNP. However, the response was still lower in KiLHRD582G mice compared to WT. Together, these data suggest that sGC activation is impaired in KiLHRD582G mice.

Figure 6
Figure 6

cGMP content in the corpora cavernosa of WT and KiLHRD582G mice. Tissues were precontracted with PE (10−5 M) and stimulated with SNP (10−5 M), ACh (10−6 M) or BAY 41-2272 (10−5 M). Data are expressed as mean ± s.e.m. (n = 5 each for basal and SNP and n = 4 each for ACh and BAY41-2272). *P < 0.05, **P < 0.01 compared to WT.

Citation: Reproduction 161, 1; 10.1530/REP-20-0447

Discussion

KiLHRD582G mice with an activating mutation in LHCGR have ED primarily due to both a loss in cavernosal smooth muscle content (Hai et al. 2017) and function. The mechanism of smooth muscle dysfunction is due to an impairment in the NO-mediated pathway of smooth muscle relaxation without a change in the Rho-kinase mediated contractile pathway. The D582G mutation in the KiLHRD582G mice is analogous to the D578G mutation found in boys with FMPP. Young adult KiLHRD582G mice are fertile for a short period of 3–4 months and develop ED as they age (Hai et al. 2017). The D578G mutation in LHCGR is germline, indicating that men harboring this mutation are not completely infertile (Shenker et al. 1993). However, there are no reports on the reproductive health of older FMPP patients. A significant finding from this study with the KiLHRD582G mouse model is that it predicts that ED is a long-term reproductive pathology in FMPP.

One of the reproductive phenotypes exhibited by KiLHRD582G male mice was the inability to successfully initiate and/or maintain penile intromission followed by ejaculation (Hai et al. 2017). These results suggested that KiLHRD582G mice had erectile dysfunction; however, it could not be confirmed as visual assessment of penile erection during the sexual behavior studies was not possible (Hai et al. 2017). Herein, the apomorphine-induced erection studies provide evidence that KiLHRD582G mice had impaired erectile function. Apomorphine is a dopamine agonist that is widely used to test erectile function in conscious animals and is the major neurotransmitter in the CNS shown to play a role in erectile function (Bernabé et al. 1999, Rampin et al. 2003, Simonsen et al. 2016). Systemic administration of apomorphine has been shown to elicit penile erections and is likely due to the involvement of D2 receptors at the central or spinal level (Giuliano & Allard 2001, Giuliano et al. 2002, Matsumoto et al. 2005, Simonsen et al. 2016).

Normal erection requires the relaxation of the cavernosal smooth muscle allowing for expansion of the sinusoids and trapping of blood by compression of the subtunical venules to reduce blood outflow (Nehra et al. 1998, Traish 2009). This veno-occlusive process requires compliance of the extracellular matrix comprised of collagen, elastic fibers and proteoglycans. This network of ECM and smooth muscle is essential for maintaining flaccidity and normal erectile response of the penis. Type I and III collagen are the major forms of collagen in the penile cavernosa responsible for high tensile strength during erection. Type I collagen is present predominantly while type III collagen is in minor proportions in the cavernosa (Raviv et al. 1997, Moreland et al. 1995). Elastic fibers in the penis are mainly composed of elastin deposited on a scaffolding of fibrillin and fibrillin-rich microfibrils and are located in sinusoids and veins in the cavernosa. Models of erectile dysfunction due to aging, diabetes or hypogonadism show a decrease in smooth muscle and increase in collagen content (Traish et al. 1999, 2003, Ferrini et al. 2007, Xie et al. 2007, Wang et al. 2015). Studies in mice and men with ED have shown a decrease in elastin and fibrillin (Sattar et al. 1994, Costa et al. 2006, Luttrell et al. 2008).

We have previously shown that KiLHRD582G mice at 6 months of age exhibit changes in the penile architecture caused by a decrease in smooth muscle content, accumulation of chondrocytes but no change in total collagen content (Hai et al. 2017). Smooth muscle loss does not appear to be due to apoptosis (data not shown) and the mechanism of chondrocyte accumulation has not been determined. Although total collagen content was unchanged, individual collagen types and elastin content were not previously determined. In this study, we demonstrated that gene expression levels of collagen I, III, elastin and fibrillin are unaltered, indicating that impairment in erectile function was primarily due to smooth muscle loss and not due to an altered ECM. However, it is possible that chondrocyte accumulation in the corpora cavernosa of the KiLHRD582G mice reduces compliance of the penis causing veno-occlusive dysfunction.

A right balance between smooth muscle contraction and relaxation is needed for normal erectile function of the penis (Morelli et al. 2006, Nunes & Webb 2012). Decreased relaxation or increased contractility of smooth muscle has been implicated in ED (Chang et al. 2003, Chitaley 2009, Jiang & Chitaley 2012, Gajbhiye et al. 2015). KiLHRD582G mice exhibited a decrease in smooth muscle contraction in response to the α1 adrenergic agonist, PE. However, the efficacy of PE, determined by EC50 values, was not different, suggesting that activation of the α1 adrenergic receptor is not altered. Because α1 adrenergic receptors are specifically localized to smooth muscle cells in the cavernosa (Traish et al. 1999) and KiLHRD582G mice have reduced cavernosal smooth muscle content (Hai et al. 2017), reduced contractile response to PE is most likely due to reduced smooth muscle content. The RhoA-Rho-kinase pathway is another mediator of smooth muscle contraction and inhibits NO-mediated smooth muscle relaxation by decreasing NOS3 expression and inhibiting NOS3 activation (Laufs & Liao 1998, Mita et al. 2005, Sugimoto et al. 2007). Our functional studies with the Rho-kinase inhibitor Y-27632 showed no change in the potency of the relaxation response indicating that this pathway was not upregulated in the KiLHRD582G mice. Normal RNA levels of the Rho-kinase pathway signaling intermediates and NOS3 further demonstrate that decreased relaxation rather than increased contractility is likely to be the cause of ED in the KiLHRD582G mice.

Endothelial cells lining the sinusoids and nonadrenergic–noncholinergic (NANC) cavernous nerves mediate smooth muscle relaxation by generation and release of NO (Dean & Lue 2005, Nunes & Webb 2012). Various animal models of aging, hypercholesterolemia and diabetes have demonstrated a significant impairment in endothelium-dependent relaxation due to a reduction in endothelial cell content, and NOS3 expression and activity in the penile cavernosa (Azadzoi & Tejada 1991, Way & Reid 1999, Burchardt et al. 2000, Akingba & Burnett 2001, Cartledge et al. 2001, Yeşilli et al. 2001, Behr-Roussel et al. 2002, Gholami et al. 2003, Liu et al. 2013). Maximal endothelium-dependent (ACh-mediated) smooth muscle relaxation was reduced in KiLHRD582 mice. Evaluation of cavernosal smooth muscle relaxation in response to the NO donor, SNP, also showed a decrease in the maximal response in KiLHRD582G mice. Because endothelial cell content and NOS3 expression levels were not altered in KiLHRD582G mice, the reduced Emax in response to both SNP and ACh is likely a reflection of decreased smooth muscle content and function in KiLHRD582G mice rather than endothelial cell loss. We were unable to perform electric field stimulation (EFS) studies to evaluate relaxation of smooth muscle by NO produced by the non-adrenergic, noncholinergic cavernous nerves. Although levels of NOS1 and NOS3 were unchanged in KiLHRD582G mice, we cannot rule out the possibility that their activities are reduced resulting in decreased endothelial and neuronal NO production that could also contribute to the reduced relaxation response in KiLHRD582G mice.

NO-mediated activation of sGC causing an increase in cGMP and cGMP-mediated activation of PRKG1 is the major mechanism for smooth muscle relaxation of the corpora cavernosa (Hedlund et al. 2000, Burnett 2006, Bivalacqua et al. 2007, Nunes & Webb 2012). cGMP levels were significantly decreased in response to ACh and SNP in KiLHRD582G mice compared to WT mice suggesting impaired activation of sGC. BAY 41-2272, which directly activates sGC independent of NO availability, significantly increased cGMP levels over that produced by ACh or SNP alone in both WT and KiLHRD582G mice. However, the levels in the KiLHRD582G mice were not rescued to the levels seen in the WT mice. Together these data suggest that the decrease in cGMP in KiLHRD582G mice is due to impaired activation of sGC.

Another potential mechanism that can also contribute to the reduced cGMP levels is its increased hydrolysis by PDE5A. However, results from this study showed lower levels of PDE5A, which is likely due to reduced smooth muscle content in the cavernosa of KiLHRD582G mice. In support of our finding, several studies have shown that expression of PDE5A is specific to smooth muscle cells and reduced expression of PDE5A is a consequence of reduction in smooth muscle content (Traish et al. 1999, 2003, 2005, Yang et al. 2009). Confirming this, a significant change in RNA levels of Pde5a was not observed when it was normalized to smooth muscle actin RNA.

At present, we do not know if this decrease in cGMP results in decreased PRKG1 activity in KiLHRD582G mice. Surprisingly, RNA and protein levels of the enzyme in the corpora cavernosa were unchanged. We expected a decrease in the PRKG1 levels as the enzyme is expressed in the cavernosal smooth muscle cells which are decreased in the KiLHRD582G mice (Hai et al. 2017). However, reports suggest that PRKG1 is also expressed in the smooth muscle of the cavernosal arteries and perhaps also the endothelial cells lining the arteries and sinusoids (Hedlund et al. 2000, Waldkirch et al. 2008). As a result, a decrease in PRKG1 due to smooth muscle loss could potentially be masked by normal levels of expression in these other cell types.

The well-documented pathologies of Leydig cell hyperplasia and adenomas seen in the testes of FMPP patients and in the KiLHRD582G mice are due to the direct action of the constitutively activated LHCGR in the Leydig cells (Shenker et al. 1993, Liu et al. 1999, Boot et al. 2011, Hai et al. 2017, McGee & Narayan 2013). In this study, we have demonstrated that constitutive activation of the LHCGR results in extragonadal pathologies. We propose that the extragonadal effects on penile smooth muscle function are indirectly mediated by testosterone rather than a direct action of LHCGR as there is no evidence for functional LHCGR in the corpus cavernosum (Hai et al. 2017). Support for this hypothesis is provided by our previous study that showed that high levels of testosterone decrease smooth muscle content in the penile corpora cavernosa and that a narrow range of optimal testosterone levels regulate penile morphology (Hiremath et al. 2020). Both low levels of testosterone, as seen in the hypogonadism, and supraphysiological levels of testosterone, as seen in FMPP, decrease penile smooth muscle content (Traish et al. 1999, Okumu et al. 2014, Wang et al. 2015, Hiremath et al. 2020). It is thought that the primary mechanism by which testosterone regulates erectile physiology is by regulating the expression and activity of NOS based on animal models of castration and testosterone replacement. Castration results in decreased NOS mRNA or protein expression and activity which are restored with testosterone replacement (Zvara et al. 1995, Penson et al. 1996, Marin et al. 1999, Park et al. 1999, Seo et al. 1999). We did not see a change in the expression of NOS1 or NOS3 in the KiLHRD582G mice compared to controls suggesting that while physiological levels of testosterone are required for NOS expression, supraphysiological levels of testosterone have no effect. However, as mentioned above, NOS activities were not determined.

In summary, the lack of apomorphine-induced erection and decreased smooth muscle relaxation provide evidence that older KiLHRD582G mice have erectile dysfunction. Impaired NO-mediated activation of sGC resulting in decreased levels of cGMP is responsible for the decreased smooth muscle relaxation. The mechanism by which sGC activation is reduced in KiLHRD582G mice requires further investigation.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported in part by the Research Seed Grant from SIU School of Medicine (P N) and an award from the DiaComp Pilot/Feasibility Program, NIDDK (R C W).

Author contribution statement

D S H conceived the study, performed experiments, analyzed and interpreted the data and wrote the manuscript. F B M P and R C W provided expert advice on the myograph experiments and analysis of the myograph data. C K assisted with the myograph studies. P N conceived and supervised the study, analyzed and interpreted the data, wrote and finalized the manuscript. All authors provided critical feedback on the manuscript.

Acknowledgements

The authors would like to thank Stacey McGee for assistance with the processing and embedding of tissues and Dr Johanna Hannan for helpful suggestions on the apomorphine studies.

References

  • Ahn GJ, Sohn YS, Kang KK, Ahn BO, Kwon JW, Kang SK, Lee BC & Hwang WS 2005 The effect of PDE5 inhibition on the erectile function in streptozotocin-induced diabetic rats. International Journal of Impotence Research 17 134141. (https://doi.org/10.1038/sj.ijir.3901295)

    • Search Google Scholar
    • Export Citation
  • Akingba AG & Burnett AL 2001 Endothelial nitric oxide synthase protein expression, localization, and activity in the penis of the alloxan-induced diabetic rat. Molecular Urology 5 189197. (https://doi.org/10.1089/10915360152745885)

    • Search Google Scholar
    • Export Citation
  • Andersson KE 2011 Mechanisms of penile erection and basis for pharmacological treatment of erectile dysfunction. Pharmacological Reviews 63 811859. (https://doi.org/10.1124/pr.111.004515)

    • Search Google Scholar
    • Export Citation
  • Ascoli M, Fanelli F & Segaloff DL 2002 The lutropin/choriogonadotropin receptor, a 2002 perspective. Endocrine Reviews 23 141174. (https://doi.org/10.1210/edrv.23.2.0462)

    • Search Google Scholar
    • Export Citation
  • Azadzoi KM & Tejada ISD 1991 Hypercholesterolemia impairs endothelium-dependent relaxation of rabbit corpus cavernosum smooth muscle. Journal of Urology 146 238240. (https://doi.org/10.1016/s0022-5347(17)37759-5)

    • Search Google Scholar
    • Export Citation
  • Behr-Roussel D, Bernabe J, Compagnie S, Rupin A, Verbeuren TJ, Alexandre L & Giuliano F 2002 Distinct mechanisms implicated in atherosclerosis-induced erectile dysfunction in rabbits. Atherosclerosis 162 355362. (https://doi.org/10.1016/s0021-9150(01)00740-7)

    • Search Google Scholar
    • Export Citation
  • Bernabé J, Rampin O, Sachs BD & Giuliano F 1999 Intracavernous pressure during erection in rats: an integrative approach based on telemetric recording. American Journal of Physiology 276 R441R449. (https://doi.org/10.1152/ajpregu.1999.276.2.R441)

    • Search Google Scholar
    • Export Citation
  • Bivalacqua TJ, Champion HC, Usta MF, Cellek S, Chitaley K, Webb RC, Lewis RL, Mills TM, Hellstrom WJ & Kadowitz PJ 2004 RhoA/Rho-kinase suppresses endothelial nitric oxide synthase in the penis: a mechanism for diabetes-associated erectile dysfunction. PNAS 101 91219126. (https://doi.org/10.1073/pnas.0400520101)

    • Search Google Scholar
    • Export Citation
  • Bivalacqua TJ, Liu T, Musicki B, Champion HC & Burnett AL 2007 Endothelial nitric oxide synthase keeps erection regulatory function balance in the penis. European Urology 51 17321740. (https://doi.org/10.1016/j.eururo.2006.10.061)

    • Search Google Scholar
    • Export Citation
  • Boot AM, Lumbroso S, Verhoef-Post M, Richter-Unruh A, Looijenga LH, Funaro A, Beishuizen A, Van Marle A, Drop SL & Themmen AP 2011 Mutation analysis of the LH receptor gene in Leydig cell adenoma and hyperplasia and functional and biochemical studies of activating mutations of the LH receptor gene. Journal of Clinical Endocrinology and Metabolism 96 E1197E1205. (https://doi.org/10.1210/jc.2010-3031)

    • Search Google Scholar
    • Export Citation
  • Burchardt T, Burchardt M, Karden J, Buttyan R, Shabsigh A, De La Taille A, Ng PY, Anastasiadis AG & Shabsigh R 2000 Reduction of endothelial and smooth muscle density in the corpora cavernosa of the streptozotocin induced diabetic rat. Journal of Urology 164 18071 81 1. (https://doi.org/10.1016/S0022-5347(05)67111-X)

    • Search Google Scholar
    • Export Citation
  • Burnett AL 2006 The role of nitric oxide in erectile dysfunction: implications for medical therapy. Journal of Clinical Hypertension 8 (Supplement 4) 5362. (https://doi.org/10.1111/j.1524-6175.2006.06026.x)

    • Search Google Scholar
    • Export Citation
  • Cartledge JJ, Eardley I & Morrison JFB 2001 Nitric oxide‐mediated corpus cavernosal smooth muscle relaxation is impaired in ageing and diabetes. BJU International 87 402407. (https://doi.org/10.1046/j.1464-410x.2001.00067.x)

    • Search Google Scholar
    • Export Citation
  • Chang S, Hypolite JA, Changolkar A, Wein AJ, Chacko S & Disanto ME 2003 Increased contractility of diabetic rabbit corpora smooth muscle in response to endothelin is mediated via Rho-kinase β. International Journal of Impotence Research 15 5362. (https://doi.org/10.1038/sj.ijir.3900947)

    • Search Google Scholar
    • Export Citation
  • Chiou WF, Liu HK & Juan CW 2010 Abnormal protein expression in the corpus cavernosum impairs erectile function in type 2 diabetes. BJU International 105 674680. (https://doi.org/10.1111/j.1464-410X.2009.08852.x)

    • Search Google Scholar
    • Export Citation
  • Chitaley K 2009 Type 1 and Type 2 diabetic‐erectile dysfunction: same diagnosis (ICD‐9), different disease? Journal of Sexual Medicine 6 (Supplement 3) 262268. (https://doi.org/10.1111/j.1743-6109.2008.01183.x)

    • Search Google Scholar
    • Export Citation
  • Costa WS, Carrerete FB, Horta WG & Sampaio FJ 2006 Comparative analysis of the penis corpora cavernosa in controls and patients with erectile dysfunction. BJU International 97 567569. (https://doi.org/10.1111/j.1464-410X.2005.05917.x)

    • Search Google Scholar
    • Export Citation
  • Dean RC & Lue TF 2005 Physiology of penile erection and pathophysiology of erectile dysfunction. Urologic Clinics of North America 32 37939 5, v. (https://doi.org/10.1016/j.ucl.2005.08.007)

    • Search Google Scholar
    • Export Citation
  • Ferrini MG, Kovanecz I, Sanchez S, Vernet D, Davila HH, Rajfer J & Gonzalez-Cadavid NF 2007 Long-term continuous treatment with sildenafil ameliorates aging-related erectile dysfunction and the underlying corporal fibrosis in the rat. Biology of Reproduction 76 9159 23. (https://doi.org/10.1095/biolreprod.106.059642)

    • Search Google Scholar
    • Export Citation
  • Gajbhiye SV, Jadhav KS, Marathe PA & Pawar DB 2015 Animal models of erectile dysfunction. Indian Journal of Urology 31 15–21. (https://doi.org/10.4103/0970-1591.128496)

    • Search Google Scholar
    • Export Citation
  • Gholami SS, Rogers R, Chang J, Ho HC, Grazziottin T, Lin CS & Lue TF 2003 The effect of vascular endothelial growth factor and adeno-associated virus mediated brain derived neurotrophic factor on neurogenic and vasculogenic erectile dysfunction induced by hyperlipidemia. Journal of Urology 169 15771581. (https://doi.org/10.1097/01.ju.0000055120.73261.76)

    • Search Google Scholar
    • Export Citation
  • Giuliano F & Allard J 2001 Dopamine and sexual function. International Journal of Impotence Research 13 ( Supplement 3) S18S28. (https://doi.org/10.1038/sj.ijir.3900719)

    • Search Google Scholar
    • Export Citation
  • Giuliano F, Allard J, Rampin O, Droupy S, Benoit G, Alexandre L & Bernabe J 2002 Pro-erectile effect of systemic apomorphine: existence of a spinal site of action. Journal of Urology 167 402406. (https://doi.org/10.1016/S0022-5347(05)65476-6)

    • Search Google Scholar
    • Export Citation
  • Hai L, Hiremath DS, Paquet M & Narayan P 2017 Constitutive luteinizing hormone receptor signaling causes sexual dysfunction and Leydig cell adenomas in male mice. Biology of Reproduction 96 10071018. (https://doi.org/10.1095/biolreprod.116.146605)

    • Search Google Scholar
    • Export Citation
  • Hedlund P, Aszódi A, Pfeifer A, Alm P, Hofmann F, Ahmad M, Fässler R & Andersson KE 2000 Erectile dysfunction in cyclic GMP-dependent kinase I-deficient mice. PNAS 97 23492354. (https://doi.org/10.1073/pnas.030419997)

    • Search Google Scholar
    • Export Citation
  • Hiremath DS, Geerling EC, Hai L & Narayan P 2020 High levels of androgens cause chondrocyte accumulation and loss of smooth muscle in the mouse penile body. Biology of Reproduction 102 12251233. (https://doi.org/10.1093/biolre/ioaa023)

    • Search Google Scholar
    • Export Citation
  • Jiang X & Chitaley K 2012 The promise of inhibition of smooth muscle tone as a treatment for erectile dysfunction: where are we now? International Journal of Impotence Research 24 4960. (https://doi.org/10.1038/ijir.2011.49)

    • Search Google Scholar
    • Export Citation
  • Laufs U & Liao JK 1998 Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. Journal of Biological Chemistry 273 2426624271. (https://doi.org/10.1074/jbc.273.37.24266)

    • Search Google Scholar
    • Export Citation
  • Liu G, Duranteau L, Carel JC, Monroe J, Doyle DA & Shenker A 1999 Leydig-cell tumors caused by an activating mutation of the gene encoding the luteinizing hormone receptor. New England Journal of Medicine 341 17311736. (https://doi.org/10.1056/NEJM199912023412304)

    • Search Google Scholar
    • Export Citation
  • Liu G, Sun X, Bian J, Wu R, Guan X, Ouyang B, Huang Y, Xiao H, Luo D & Atala A et al.2013 Correction of diabetic erectile dysfunction with adipose derived stem cells modified with the vascular endothelial growth factor gene in a rodent diabetic model. PLoS ONE 8 e72790. (https://doi.org/10.1371/journal.pone.0072790)

    • Search Google Scholar
    • Export Citation
  • Lombo C, Morgado C, Tavares I & Neves D 2016 Effects of prolonged ingestion of epigallocatechin gallate on diabetes type 1-induced vascular modifications in the erectile tissue of rats. International Journal of Impotence Research 28 133138. (https://doi.org/10.1038/ijir.2016.19)

    • Search Google Scholar
    • Export Citation
  • Luttrell IP, Swee M, Starcher B, Parks WC & Chitaley K 2008 Erectile dysfunction in the type II diabetic db/db mouse: impaired venoocclusion with altered cavernosal vasoreactivity and matrix. American Journal of Physiology: Heart and Circulatory Physiology 294 H2204H2211. (https://doi.org/10.1152/ajpheart.00027.2008)

    • Search Google Scholar
    • Export Citation
  • Marin R, Escrig A, Abreu P & Mas M 1999 Androgen-dependent nitric oxide release in rat penis correlates with levels of constitutive nitric oxide synthase isoenzymes. Biology of Reproduction 61 10121016. (https://doi.org/10.1095/biolreprod61.4.1012)

    • Search Google Scholar
    • Export Citation
  • Matsumoto K, Yoshida M, Andersson KE & Hedlund P 2005 Effects in vitro and in vivo by apomorphine in the rat corpus cavernosum. British Journal of Pharmacology 146 259267. (https://doi.org/10.1038/sj.bjp.0706317)

    • Search Google Scholar
    • Export Citation
  • McGee SR & Narayan P 2013 Precocious puberty and Leydig cell hyperplasia in male mice with a gain of function mutation in the LH receptor gene. Endocrinology 154 39003913. (https://doi.org/10.1210/en.2012-2179)

    • Search Google Scholar
    • Export Citation
  • Mita S-I, Kobayashi N, Yoshida K, Nakano S & Matsuoka H 2005 Cardioprotective mechanisms of Rho-kinase inhibition associated with eNOS and oxidative stress-LOX-1 pathway in Dahl salt-sensitive hypertensive rats. Journal of Hypertension 23 8796. (https://doi.org/10.1097/00004872-200501000-00017)

    • Search Google Scholar
    • Export Citation
  • Moreland RB, Traish A, Mcmillin MA, Smith B, Goldstein I & De Tejada Saenz I 1995 PGE1 suppresses the induction of collagen synthesis by transforming growth factor-beta 1 in human corpus cavernosum smooth muscle. Journal of Urology 153 826834. (https://doi.org/10.1016/S0022-5347(01)67730-9)

    • Search Google Scholar
    • Export Citation
  • Morelli A, Filippi S, Vignozzi L, Mancina R & Maggi M 2006 Physiology of erectile function: an update on intracellular molecular processes. EAU-EBU Update Series 4 96108. (https://doi.org/10.1016/j.eeus.2006.03.003)

    • Search Google Scholar
    • Export Citation
  • Mostafa ME, Senbel AM & Mostafa T 2013 Effect of chronic low-dose tadalafil on penile cavernous tissues in diabetic rats. Urology 81 12531259. (https://doi.org/10.1016/j.urology.2012.12.068)

    • Search Google Scholar
    • Export Citation
  • Narayan P, Ulloa-Aguirre A & Dias JA 2019 Gonadotropin hormones and their receptors. In Yen and Jaffe’s Reproductive Endocrinology, 8th ed. , pp. 2557. Eds Strauss RL Barbieri JLPhiladelphia, PA: Elsevier.

    • Search Google Scholar
    • Export Citation
  • Nehra A, Azadzoi KM, Moreland RB, Pabby A, Siroky MB, Krane RJ, Goldstein I & Udelson D 1998 Cavernosal expandability is an erectile tissue mechanical property which predicts trabecular histology in an animal model of vasculogenic erectile dysfunction. Journal of Urology 159 22292236. (https://doi.org/10.1016/S0022-5347(01)63311-1)

    • Search Google Scholar
    • Export Citation
  • Nunes KP & Webb RC 2012 Mechanisms in erectile function and dysfunction: an overview. In Erectile Dysfunction-Disease-Associated Mechanisms and Novel Insights into Therapy. IntechOpen.

    • Search Google Scholar
    • Export Citation
  • Okumu LA, Braden TD, Vail K, Simon L & Goyal HO 2014 Low androgen induced penile maldevelopment involves altered gene expression of biomarkers of smooth muscle differentiation and a key enzyme regulating cavernous smooth muscle cell tone. Journal of Urology 192 267273. (https://doi.org/10.1016/j.juro.2013.11.101)

    • Search Google Scholar
    • Export Citation
  • Park KH, Kim SW, Kim KD & Paick JS 1999 Effects of androgens on the expression of nitric oxide synthase mRNAs in rat corpus cavernosum. BJU International 83 327333. (https://doi.org/10.1046/j.1464-410x.1999.00913.x)

    • Search Google Scholar
    • Export Citation
  • Penson DF, Ng C, Cai L, Rajfer J & Gonzalez-Cadavid NF 1996 Androgen and pituitary control of penile nitric oxide synthase and erectile function in the rat. Biology of Reproduction 55 5675 74. (https://doi.org/10.1095/biolreprod55.3.567)

    • Search Google Scholar
    • Export Citation
  • Rampin O, Jérôme N & Suaudeau C 2003 Proerectile effects of apomorphine in mice. Life Sciences 72 23292336. (https://doi.org/10.1016/s0024-3205(03)00122-x)

    • Search Google Scholar
    • Export Citation
  • Raviv G, Kiss R, Vanegas JP, Petein M, Danguy A, Schulman C & Wespes E 1997 Objective measurement of the different collagen types in the corpus cavernosum of potent and impotent men: an immunohistochemical staining with computerized-image analysis. World Journal of Urology 15 5055. (https://doi.org/10.1007/BF01275157)

    • Search Google Scholar
    • Export Citation
  • Sattar AA, Wespes E & Schulman CC 1994 Computerized measurement of penile elastic fibres in potent and impotent men. European Urology 25 142144. (https://doi.org/10.1159/000475269)

    • Search Google Scholar
    • Export Citation
  • Seo SI, Kim SW & Paick JS 1999 The effects of androgen on penile reflex, erectile response to electrical stimulation and penile NOS activity in the rat. Asian Journal of Andrology 1 1691 74.

    • Search Google Scholar
    • Export Citation
  • Shenker A, Laue L, Kosugi S, Merendino JJ Jr, Minegishi T & Cutler Jr GB 1993 A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature 365 652–654. (https://doi.org/10.1038/365652a0)

    • Search Google Scholar
    • Export Citation
  • Simonsen U, Comerma‐Steffensen S & Andersson KE 2016 Modulation of dopaminergic pathways to treat erectile dysfunction. Basic and Clinical Pharmacology and Toxicology 119 (Supplement 3) 6374. (https://doi.org/10.1111/bcpt.12653)

    • Search Google Scholar
    • Export Citation
  • Sopko NA, Hannan JL & Bivalacqua TJ 2014 Understanding and targeting the Rho kinase pathway in erectile dysfunction. Nature Reviews: Urology 11 622–628. (https://doi.org/10.1038/nrurol.2014.278)

    • Search Google Scholar
    • Export Citation
  • Sugimoto M, Nakayama M, Goto TM, Amano M, Komori K & Kaibuchi K 2007 Rho-kinase phosphorylates eNOS at threonine 495 in endothelial cells. Biochemical and Biophysical Research Communications 361 462467. (https://doi.org/10.1016/j.bbrc.2007.07.030)

    • Search Google Scholar
    • Export Citation
  • Themmen APN & Huhtaniemi IT 2000 Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function. Endocrine Reviews 21 551583. (https://doi.org/10.1210/edrv.21.5.0409)

    • Search Google Scholar
    • Export Citation
  • Toque HA, Nunes KP, Yao L, Liao JK, Webb RC, Caldwell RB & Caldwell RW 2013 Activated Rho kinase mediates diabetes‐induced elevation of vascular arginase activation and contributes to impaired corpora cavernosa relaxation: possible involvement of p38 MAPK activation. Journal of Sexual Medicine 10 15021515. (https://doi.org/10.1111/jsm.12134)

    • Search Google Scholar
    • Export Citation
  • Traish AM 2009 Androgens play a pivotal role in maintaining penile tissue architecture and erection: a review. Journal of Andrology 30 363369. (https://doi.org/10.2164/jandrol.108.006007)

    • Search Google Scholar
    • Export Citation
  • Traish AM, Park K, Dhir V, Kim NN, Moreland RB & Goldstein I 1999 Effects of castration and androgen replacement on erectile function in a rabbit model. Endocrinology 140 18611868. (https://doi.org/10.1210/endo.140.4.6655)

    • Search Google Scholar
    • Export Citation
  • Traish A, Kim NN, Moreland RB & Goldstein I 2000 Role of alpha adrenergic receptors in erectile function. International Journal of Impotence Research 12 (Supplement48) S48S63. (https://doi.org/10.1038/sj.ijir.3900506)

    • Search Google Scholar
    • Export Citation
  • Traish AM, Munarriz R, O'connell L, Choi S, Kim SW, Kim NN, Huang YH & Goldstein I 2003 Effects of medical or surgical castration on erectile function in an animal model. Journal of Andrology 24 381387. (https://doi.org/10.1002/j.1939-4640.2003.tb02686.x)

    • Search Google Scholar
    • Export Citation
  • Traish AM, Toselli P, Jeong SJ & Kim NN 2005 Adipocyte accumulation in penile corpus cavernosum of the orchiectomized rabbit: a potential mechanism for veno‐occlusive dysfunction in androgen deficiency. Journal of Andrology 26 242248. (https://doi.org/10.1002/j.1939-4640.2005.tb01091.x)

    • Search Google Scholar
    • Export Citation
  • Ulloa-Aguirre A, Reiter E, Bousfield G, Dias JA & Huhtaniemi I 2014 Constitutive activity in gonadotropin receptors. Advances in Pharmacology 70 3780. (https://doi.org/10.1016/B978-0-12-417197-8.00002-X)

    • Search Google Scholar
    • Export Citation
  • Waldkirch E, Uckert S, Sigl K, Imkamp F, Langnaese K, Richter K, Jonas U, Sohn M, Stief C & Wolf G et al.2008 Expression and distribution of cyclic GMP-dependent protein kinase-1 isoforms in human penile erectile tissue. Journal of Sexual Medicine 5 5365 43. (https://doi.org/10.1111/j.1743-6109.2007.00735.x)

    • Search Google Scholar
    • Export Citation
  • Wang XJ, Xu TY, Xia LL, Zhong S, Zhang XH, Zhu ZW, Chen DR, Liu Y, Fan Y & Xu C et al.2015 Castration impairs erectile organ structure and function by inhibiting autophagy and promoting apoptosis of corpus cavernosum smooth muscle cells in rats. International Urology and Nephrology 47 11051115. (https://doi.org/10.1007/s11255-015-1011-3)

    • Search Google Scholar
    • Export Citation
  • Way KJ & Reid JJ 1999 The effects of diabetes on nitric oxide-mediated responses in rat corpus cavernosum. European Journal of Pharmacology 376 7382. (https://doi.org/10.1016/s0014-2999(99)00347-7)

    • Search Google Scholar
    • Export Citation
  • Xie D, Odronic SI, Wu F, Pippen A, Donatucci CF & Annex BH 2007 Mouse model of erectile dysfunction due to diet-induced diabetes mellitus. Urology 70 196201. (https://doi.org/10.1016/j.urology.2007.02.060)

    • Search Google Scholar
    • Export Citation
  • Yang R, Huang YC, Lin G, Wang G, Hung S, Dai YT, Sun ZY, Lue TF & Lin CS 2009 Lack of direct androgen regulation of PDE5 expression. Biochemical and Biophysical Research Communications 380 758762. (https://doi.org/10.1016/j.bbrc.2009.01.144)

    • Search Google Scholar
    • Export Citation
  • Yeşilli C, Yaman O & Anafarta K 2001 Effect of experimental hypercholesterolemia on cavernosal structures. Urology 57 11841188. (https://doi.org/10.1016/s0090-4295(01)00974-8)

    • Search Google Scholar
    • Export Citation
  • Zvara P, Sioufi R, Schipper HM, Begin LR & Brock GB 1995 Nitric oxide mediated erectile activity is a testosterone dependent event: a rat erection model. International Journal of Impotence Research 7 209219.

    • Search Google Scholar
    • Export Citation

 

  • Collapse
  • Expand

     An official journal of

    Society for Reproduction and Fertility

 

  • View in gallery
    Figure 1

    Normal collagen, elastin and fibrillin RNA levels in the cavernosa of KiLHRD582G mice. Relative expression of Col1a1, Col1a2, Col3a1, Eln, Fbn1 and Fbn2 represents levels of RNA normalized to internal control Rps2 and relative to calibrator sample prepared by mixing equal amounts of cDNA from all samples. Data are expressed as mean ± s.e.m. (n = 5–6).

  • View in gallery
    Figure 2

    Normal endothelium content in the cavernosa of KiLHRD582G mice. (A) Representative photomicrographs of immunohistochemical staining of MECA-32 in WT (n = 4) and KiLHRD582G (n = 6) mice. Scale bars, 100 µm. (B) Quantification of MECA-32 stained area in the corpora cavernosa of WT and KiLHRD582G mice determined by Image-J analysis. (C) Quantification of Nos3 by RT-qPCR (n = 5). Relative expression represents levels of RNA normalized to internal control Rps2 and relative to calibrator. (D) Western blot image for NOS3 protein levels. GAPDH was used as the loading control. M, Molecular weight markers, lanes 1, 3, 5, 7 – WT and lanes 2, 4, 6, 8 – KiLHRD582G (n = 4). Expected size of NOS3 is 140 kDa. (E) Levels of NOS3 relative to GAPDH was determined by quantification of intensities of bands on Western blots. (F) Concentration-dependent relaxation response induced by ACh (n = 6). Data are expressed as mean ± s.e.m. ***P < 0.001 compared to WT.

  • View in gallery
    Figure 3

    Smooth muscle dysfunction in the cavernosal segments of WT and KiLHRD582G mice. (A) Concentration-dependent contractile response induced by PE. (B) Concentration-dependent relaxation response induced by SNP. Data are expressed as mean ± s.e.m. (n = 6). ***P < 0.001, ****P < 0.0001 compared to WT.

  • View in gallery
    Figure 4

    Normal RNA and protein levels of signaling intermediates in the NO signaling pathway for smooth muscle relaxation in the cavernosa of KiLHRD582G mice. Quantification of Nos1 (A), Pde5a (C) and Prkg1 (E) by RT-qPCR (n = 5). Representative Western blot images for NOS1 (B), PDE5 (D), PRKG1 (F) protein levels. GAPDH was used as the loading control. Quantification of the level of each protein relative to GAPDH was determined by quantification of intensities of bands on Western blots (n = 4). M, molecular weight markers. Expected sizes of NOS1, PDE5, PRKG1 and GAPDH are 160, 100, 78 and 37 kDa, respectively. Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01 compared to WT.

  • View in gallery
    Figure 5

    Normal gene expression levels of signaling intermediates of the Rho-kinase smooth muscle contractile pathway in the cavernosa of KiLHRD582G mice. (A) Relaxation response induced by Y-27632, a Rho-kinase inhibitor (n = 6). Quantification of Rhoa (B), Rock1 (C), Rock2 (D) RNA by RT-qPCR (n = 5). Relative expression represents levels of RNA normalized to the internal control Rps2 and relative to the calibrator sample prepared by mixing equal amounts of cDNA from all samples. Data are expressed as mean ± s.e.m. **P < 0.01 compared to WT.

  • View in gallery
    Figure 6

    cGMP content in the corpora cavernosa of WT and KiLHRD582G mice. Tissues were precontracted with PE (10−5 M) and stimulated with SNP (10−5 M), ACh (10−6 M) or BAY 41-2272 (10−5 M). Data are expressed as mean ± s.e.m. (n = 5 each for basal and SNP and n = 4 each for ACh and BAY41-2272). *P < 0.05, **P < 0.01 compared to WT.

  • Ahn GJ, Sohn YS, Kang KK, Ahn BO, Kwon JW, Kang SK, Lee BC & Hwang WS 2005 The effect of PDE5 inhibition on the erectile function in streptozotocin-induced diabetic rats. International Journal of Impotence Research 17 134141. (https://doi.org/10.1038/sj.ijir.3901295)

    • Search Google Scholar
    • Export Citation
  • Akingba AG & Burnett AL 2001 Endothelial nitric oxide synthase protein expression, localization, and activity in the penis of the alloxan-induced diabetic rat. Molecular Urology 5 189197. (https://doi.org/10.1089/10915360152745885)

    • Search Google Scholar
    • Export Citation
  • Andersson KE 2011 Mechanisms of penile erection and basis for pharmacological treatment of erectile dysfunction. Pharmacological Reviews 63 811859. (https://doi.org/10.1124/pr.111.004515)

    • Search Google Scholar
    • Export Citation
  • Ascoli M, Fanelli F & Segaloff DL 2002 The lutropin/choriogonadotropin receptor, a 2002 perspective. Endocrine Reviews 23 141174. (https://doi.org/10.1210/edrv.23.2.0462)

    • Search Google Scholar
    • Export Citation
  • Azadzoi KM & Tejada ISD 1991 Hypercholesterolemia impairs endothelium-dependent relaxation of rabbit corpus cavernosum smooth muscle. Journal of Urology 146 238240. (https://doi.org/10.1016/s0022-5347(17)37759-5)

    • Search Google Scholar
    • Export Citation
  • Behr-Roussel D, Bernabe J, Compagnie S, Rupin A, Verbeuren TJ, Alexandre L & Giuliano F 2002 Distinct mechanisms implicated in atherosclerosis-induced erectile dysfunction in rabbits. Atherosclerosis 162 355362. (https://doi.org/10.1016/s0021-9150(01)00740-7)

    • Search Google Scholar
    • Export Citation
  • Bernabé J, Rampin O, Sachs BD & Giuliano F 1999 Intracavernous pressure during erection in rats: an integrative approach based on telemetric recording. American Journal of Physiology 276 R441R449. (https://doi.org/10.1152/ajpregu.1999.276.2.R441)

    • Search Google Scholar
    • Export Citation
  • Bivalacqua TJ, Champion HC, Usta MF, Cellek S, Chitaley K, Webb RC, Lewis RL, Mills TM, Hellstrom WJ & Kadowitz PJ 2004 RhoA/Rho-kinase suppresses endothelial nitric oxide synthase in the penis: a mechanism for diabetes-associated erectile dysfunction. PNAS 101 91219126. (https://doi.org/10.1073/pnas.0400520101)

    • Search Google Scholar
    • Export Citation
  • Bivalacqua TJ, Liu T, Musicki B, Champion HC & Burnett AL 2007 Endothelial nitric oxide synthase keeps erection regulatory function balance in the penis. European Urology 51 17321740. (https://doi.org/10.1016/j.eururo.2006.10.061)

    • Search Google Scholar
    • Export Citation
  • Boot AM, Lumbroso S, Verhoef-Post M, Richter-Unruh A, Looijenga LH, Funaro A, Beishuizen A, Van Marle A, Drop SL & Themmen AP 2011 Mutation analysis of the LH receptor gene in Leydig cell adenoma and hyperplasia and functional and biochemical studies of activating mutations of the LH receptor gene. Journal of Clinical Endocrinology and Metabolism 96 E1197E1205. (https://doi.org/10.1210/jc.2010-3031)

    • Search Google Scholar
    • Export Citation
  • Burchardt T, Burchardt M, Karden J, Buttyan R, Shabsigh A, De La Taille A, Ng PY, Anastasiadis AG & Shabsigh R 2000 Reduction of endothelial and smooth muscle density in the corpora cavernosa of the streptozotocin induced diabetic rat. Journal of Urology 164 18071 81 1. (https://doi.org/10.1016/S0022-5347(05)67111-X)

    • Search Google Scholar
    • Export Citation
  • Burnett AL 2006 The role of nitric oxide in erectile dysfunction: implications for medical therapy. Journal of Clinical Hypertension 8 (Supplement 4) 5362. (https://doi.org/10.1111/j.1524-6175.2006.06026.x)

    • Search Google Scholar
    • Export Citation
  • Cartledge JJ, Eardley I & Morrison JFB 2001 Nitric oxide‐mediated corpus cavernosal smooth muscle relaxation is impaired in ageing and diabetes. BJU International 87 402407. (https://doi.org/10.1046/j.1464-410x.2001.00067.x)

    • Search Google Scholar
    • Export Citation
  • Chang S, Hypolite JA, Changolkar A, Wein AJ, Chacko S & Disanto ME 2003 Increased contractility of diabetic rabbit corpora smooth muscle in response to endothelin is mediated via Rho-kinase β. International Journal of Impotence Research 15 5362. (https://doi.org/10.1038/sj.ijir.3900947)

    • Search Google Scholar
    • Export Citation
  • Chiou WF, Liu HK & Juan CW 2010 Abnormal protein expression in the corpus cavernosum impairs erectile function in type 2 diabetes. BJU International 105 674680. (https://doi.org/10.1111/j.1464-410X.2009.08852.x)

    • Search Google Scholar
    • Export Citation
  • Chitaley K 2009 Type 1 and Type 2 diabetic‐erectile dysfunction: same diagnosis (ICD‐9), different disease? Journal of Sexual Medicine 6 (Supplement 3) 262268. (https://doi.org/10.1111/j.1743-6109.2008.01183.x)

    • Search Google Scholar
    • Export Citation
  • Costa WS, Carrerete FB, Horta WG & Sampaio FJ 2006 Comparative analysis of the penis corpora cavernosa in controls and patients with erectile dysfunction. BJU International 97 567569. (https://doi.org/10.1111/j.1464-410X.2005.05917.x)

    • Search Google Scholar
    • Export Citation
  • Dean RC & Lue TF 2005 Physiology of penile erection and pathophysiology of erectile dysfunction. Urologic Clinics of North America 32 37939 5, v. (https://doi.org/10.1016/j.ucl.2005.08.007)

    • Search Google Scholar
    • Export Citation
  • Ferrini MG, Kovanecz I, Sanchez S, Vernet D, Davila HH, Rajfer J & Gonzalez-Cadavid NF 2007 Long-term continuous treatment with sildenafil ameliorates aging-related erectile dysfunction and the underlying corporal fibrosis in the rat. Biology of Reproduction 76 9159 23. (https://doi.org/10.1095/biolreprod.106.059642)

    • Search Google Scholar
    • Export Citation
  • Gajbhiye SV, Jadhav KS, Marathe PA & Pawar DB 2015 Animal models of erectile dysfunction. Indian Journal of Urology 31 15–21. (https://doi.org/10.4103/0970-1591.128496)

    • Search Google Scholar
    • Export Citation
  • Gholami SS, Rogers R, Chang J, Ho HC, Grazziottin T, Lin CS & Lue TF 2003 The effect of vascular endothelial growth factor and adeno-associated virus mediated brain derived neurotrophic factor on neurogenic and vasculogenic erectile dysfunction induced by hyperlipidemia. Journal of Urology 169 15771581. (https://doi.org/10.1097/01.ju.0000055120.73261.76)

    • Search Google Scholar
    • Export Citation
  • Giuliano F & Allard J 2001 Dopamine and sexual function. International Journal of Impotence Research 13 ( Supplement 3) S18S28. (https://doi.org/10.1038/sj.ijir.3900719)

    • Search Google Scholar
    • Export Citation
  • Giuliano F, Allard J, Rampin O, Droupy S, Benoit G, Alexandre L & Bernabe J 2002 Pro-erectile effect of systemic apomorphine: existence of a spinal site of action. Journal of Urology 167 402406. (https://doi.org/10.1016/S0022-5347(05)65476-6)

    • Search Google Scholar
    • Export Citation
  • Hai L, Hiremath DS, Paquet M & Narayan P 2017 Constitutive luteinizing hormone receptor signaling causes sexual dysfunction and Leydig cell adenomas in male mice. Biology of Reproduction 96 10071018. (https://doi.org/10.1095/biolreprod.116.146605)

    • Search Google Scholar
    • Export Citation
  • Hedlund P, Aszódi A, Pfeifer A, Alm P, Hofmann F, Ahmad M, Fässler R & Andersson KE 2000 Erectile dysfunction in cyclic GMP-dependent kinase I-deficient mice. PNAS 97 23492354. (https://doi.org/10.1073/pnas.030419997)

    • Search Google Scholar
    • Export Citation
  • Hiremath DS, Geerling EC, Hai L & Narayan P 2020 High levels of androgens cause chondrocyte accumulation and loss of smooth muscle in the mouse penile body. Biology of Reproduction 102 12251233. (https://doi.org/10.1093/biolre/ioaa023)

    • Search Google Scholar
    • Export Citation
  • Jiang X & Chitaley K 2012 The promise of inhibition of smooth muscle tone as a treatment for erectile dysfunction: where are we now? International Journal of Impotence Research 24 4960. (https://doi.org/10.1038/ijir.2011.49)

    • Search Google Scholar
    • Export Citation
  • Laufs U & Liao JK 1998 Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. Journal of Biological Chemistry 273 2426624271. (https://doi.org/10.1074/jbc.273.37.24266)

    • Search Google Scholar
    • Export Citation
  • Liu G, Duranteau L, Carel JC, Monroe J, Doyle DA & Shenker A 1999 Leydig-cell tumors caused by an activating mutation of the gene encoding the luteinizing hormone receptor. New England Journal of Medicine 341 17311736. (https://doi.org/10.1056/NEJM199912023412304)

    • Search Google Scholar
    • Export Citation
  • Liu G, Sun X, Bian J, Wu R, Guan X, Ouyang B, Huang Y, Xiao H, Luo D & Atala A et al.2013 Correction of diabetic erectile dysfunction with adipose derived stem cells modified with the vascular endothelial growth factor gene in a rodent diabetic model. PLoS ONE 8 e72790. (https://doi.org/10.1371/journal.pone.0072790)

    • Search Google Scholar
    • Export Citation
  • Lombo C, Morgado C, Tavares I & Neves D 2016 Effects of prolonged ingestion of epigallocatechin gallate on diabetes type 1-induced vascular modifications in the erectile tissue of rats. International Journal of Impotence Research 28 133138. (https://doi.org/10.1038/ijir.2016.19)

    • Search Google Scholar
    • Export Citation
  • Luttrell IP, Swee M, Starcher B, Parks WC & Chitaley K 2008 Erectile dysfunction in the type II diabetic db/db mouse: impaired venoocclusion with altered cavernosal vasoreactivity and matrix. American Journal of Physiology: Heart and Circulatory Physiology 294 H2204H2211. (https://doi.org/10.1152/ajpheart.00027.2008)

    • Search Google Scholar
    • Export Citation
  • Marin R, Escrig A, Abreu P & Mas M 1999 Androgen-dependent nitric oxide release in rat penis correlates with levels of constitutive nitric oxide synthase isoenzymes. Biology of Reproduction 61 10121016. (https://doi.org/10.1095/biolreprod61.4.1012)

    • Search Google Scholar
    • Export Citation
  • Matsumoto K, Yoshida M, Andersson KE & Hedlund P 2005 Effects in vitro and in vivo by apomorphine in the rat corpus cavernosum. British Journal of Pharmacology 146 259267. (https://doi.org/10.1038/sj.bjp.0706317)

    • Search Google Scholar
    • Export Citation
  • McGee SR & Narayan P 2013 Precocious puberty and Leydig cell hyperplasia in male mice with a gain of function mutation in the LH receptor gene. Endocrinology 154 39003913. (https://doi.org/10.1210/en.2012-2179)

    • Search Google Scholar
    • Export Citation
  • Mita S-I, Kobayashi N, Yoshida K, Nakano S & Matsuoka H 2005 Cardioprotective mechanisms of Rho-kinase inhibition associated with eNOS and oxidative stress-LOX-1 pathway in Dahl salt-sensitive hypertensive rats. Journal of Hypertension 23 8796. (https://doi.org/10.1097/00004872-200501000-00017)

    • Search Google Scholar
    • Export Citation
  • Moreland RB, Traish A, Mcmillin MA, Smith B, Goldstein I & De Tejada Saenz I 1995 PGE1 suppresses the induction of collagen synthesis by transforming growth factor-beta 1 in human corpus cavernosum smooth muscle. Journal of Urology 153 826834. (https://doi.org/10.1016/S0022-5347(01)67730-9)

    • Search Google Scholar
    • Export Citation
  • Morelli A, Filippi S, Vignozzi L, Mancina R & Maggi M 2006 Physiology of erectile function: an update on intracellular molecular processes. EAU-EBU Update Series 4 96108. (https://doi.org/10.1016/j.eeus.2006.03.003)

    • Search Google Scholar
    • Export Citation
  • Mostafa ME, Senbel AM & Mostafa T 2013 Effect of chronic low-dose tadalafil on penile cavernous tissues in diabetic rats. Urology 81 12531259. (https://doi.org/10.1016/j.urology.2012.12.068)

    • Search Google Scholar
    • Export Citation
  • Narayan P, Ulloa-Aguirre A & Dias JA 2019 Gonadotropin hormones and their receptors. In Yen and Jaffe’s Reproductive Endocrinology, 8th ed. , pp. 2557. Eds Strauss RL Barbieri JLPhiladelphia, PA: Elsevier.

    • Search Google Scholar
    • Export Citation
  • Nehra A, Azadzoi KM, Moreland RB, Pabby A, Siroky MB, Krane RJ, Goldstein I & Udelson D 1998 Cavernosal expandability is an erectile tissue mechanical property which predicts trabecular histology in an animal model of vasculogenic erectile dysfunction. Journal of Urology 159 22292236. (https://doi.org/10.1016/S0022-5347(01)63311-1)

    • Search Google Scholar
    • Export Citation
  • Nunes KP & Webb RC 2012 Mechanisms in erectile function and dysfunction: an overview. In Erectile Dysfunction-Disease-Associated Mechanisms and Novel Insights into Therapy. IntechOpen.

    • Search Google Scholar
    • Export Citation
  • Okumu LA, Braden TD, Vail K, Simon L & Goyal HO 2014 Low androgen induced penile maldevelopment involves altered gene expression of biomarkers of smooth muscle differentiation and a key enzyme regulating cavernous smooth muscle cell tone. Journal of Urology 192 267273. (https://doi.org/10.1016/j.juro.2013.11.101)

    • Search Google Scholar
    • Export Citation
  • Park KH, Kim SW, Kim KD & Paick JS 1999 Effects of androgens on the expression of nitric oxide synthase mRNAs in rat corpus cavernosum. BJU International 83 327333. (https://doi.org/10.1046/j.1464-410x.1999.00913.x)

    • Search Google Scholar
    • Export Citation
  • Penson DF, Ng C, Cai L, Rajfer J & Gonzalez-Cadavid NF 1996 Androgen and pituitary control of penile nitric oxide synthase and erectile function in the rat. Biology of Reproduction 55 5675 74. (https://doi.org/10.1095/biolreprod55.3.567)

    • Search Google Scholar
    • Export Citation
  • Rampin O, Jérôme N & Suaudeau C 2003 Proerectile effects of apomorphine in mice. Life Sciences 72 23292336. (https://doi.org/10.1016/s0024-3205(03)00122-x)

    • Search Google Scholar
    • Export Citation
  • Raviv G, Kiss R, Vanegas JP, Petein M, Danguy A, Schulman C & Wespes E 1997 Objective measurement of the different collagen types in the corpus cavernosum of potent and impotent men: an immunohistochemical staining with computerized-image analysis. World Journal of Urology 15 5055. (https://doi.org/10.1007/BF01275157)

    • Search Google Scholar
    • Export Citation
  • Sattar AA, Wespes E & Schulman CC 1994 Computerized measurement of penile elastic fibres in potent and impotent men. European Urology 25 142144. (https://doi.org/10.1159/000475269)

    • Search Google Scholar
    • Export Citation
  • Seo SI, Kim SW & Paick JS 1999 The effects of androgen on penile reflex, erectile response to electrical stimulation and penile NOS activity in the rat. Asian Journal of Andrology 1 1691 74.

    • Search Google Scholar
    • Export Citation
  • Shenker A, Laue L, Kosugi S, Merendino JJ Jr, Minegishi T & Cutler Jr GB 1993 A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature 365 652–654. (https://doi.org/10.1038/365652a0)

    • Search Google Scholar
    • Export Citation
  • Simonsen U, Comerma‐Steffensen S & Andersson KE 2016 Modulation of dopaminergic pathways to treat erectile dysfunction. Basic and Clinical Pharmacology and Toxicology 119 (Supplement 3) 6374. (https://doi.org/10.1111/bcpt.12653)

    • Search Google Scholar
    • Export Citation
  • Sopko NA, Hannan JL & Bivalacqua TJ 2014 Understanding and targeting the Rho kinase pathway in erectile dysfunction. Nature Reviews: Urology 11 622–628. (https://doi.org/10.1038/nrurol.2014.278)

    • Search Google Scholar
    • Export Citation
  • Sugimoto M, Nakayama M, Goto TM, Amano M, Komori K & Kaibuchi K 2007 Rho-kinase phosphorylates eNOS at threonine 495 in endothelial cells. Biochemical and Biophysical Research Communications 361 462467. (https://doi.org/10.1016/j.bbrc.2007.07.030)

    • Search Google Scholar
    • Export Citation
  • Themmen APN & Huhtaniemi IT 2000 Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function. Endocrine Reviews 21 551583. (https://doi.org/10.1210/edrv.21.5.0409)

    • Search Google Scholar
    • Export Citation
  • Toque HA, Nunes KP, Yao L, Liao JK, Webb RC, Caldwell RB & Caldwell RW 2013 Activated Rho kinase mediates diabetes‐induced elevation of vascular arginase activation and contributes to impaired corpora cavernosa relaxation: possible involvement of p38 MAPK activation. Journal of Sexual Medicine 10 15021515. (https://doi.org/10.1111/jsm.12134)

    • Search Google Scholar
    • Export Citation
  • Traish AM 2009 Androgens play a pivotal role in maintaining penile tissue architecture and erection: a review. Journal of Andrology 30 363369. (https://doi.org/10.2164/jandrol.108.006007)

    • Search Google Scholar
    • Export Citation
  • Traish AM, Park K, Dhir V, Kim NN, Moreland RB & Goldstein I 1999 Effects of castration and androgen replacement on erectile function in a rabbit model. Endocrinology 140 18611868. (https://doi.org/10.1210/endo.140.4.6655)

    • Search Google Scholar
    • Export Citation
  • Traish A, Kim NN, Moreland RB & Goldstein I 2000 Role of alpha adrenergic receptors in erectile function. International Journal of Impotence Research 12 (Supplement48) S48S63. (https://doi.org/10.1038/sj.ijir.3900506)

    • Search Google Scholar
    • Export Citation
  • Traish AM, Munarriz R, O'connell L, Choi S, Kim SW, Kim NN, Huang YH & Goldstein I 2003 Effects of medical or surgical castration on erectile function in an animal model. Journal of Andrology 24 381387. (https://doi.org/10.1002/j.1939-4640.2003.tb02686.x)

    • Search Google Scholar
    • Export Citation
  • Traish AM, Toselli P, Jeong SJ & Kim NN 2005 Adipocyte accumulation in penile corpus cavernosum of the orchiectomized rabbit: a potential mechanism for veno‐occlusive dysfunction in androgen deficiency. Journal of Andrology 26 242248. (https://doi.org/10.1002/j.1939-4640.2005.tb01091.x)

    • Search Google Scholar
    • Export Citation
  • Ulloa-Aguirre A, Reiter E, Bousfield G, Dias JA & Huhtaniemi I 2014 Constitutive activity in gonadotropin receptors. Advances in Pharmacology 70 3780. (https://doi.org/10.1016/B978-0-12-417197-8.00002-X)

    • Search Google Scholar
    • Export Citation
  • Waldkirch E, Uckert S, Sigl K, Imkamp F, Langnaese K, Richter K, Jonas U, Sohn M, Stief C & Wolf G et al.2008 Expression and distribution of cyclic GMP-dependent protein kinase-1 isoforms in human penile erectile tissue. Journal of Sexual Medicine 5 5365 43. (https://doi.org/10.1111/j.1743-6109.2007.00735.x)

    • Search Google Scholar
    • Export Citation
  • Wang XJ, Xu TY, Xia LL, Zhong S, Zhang XH, Zhu ZW, Chen DR, Liu Y, Fan Y & Xu C et al.2015 Castration impairs erectile organ structure and function by inhibiting autophagy and promoting apoptosis of corpus cavernosum smooth muscle cells in rats. International Urology and Nephrology 47 11051115. (https://doi.org/10.1007/s11255-015-1011-3)

    • Search Google Scholar
    • Export Citation
  • Way KJ & Reid JJ 1999 The effects of diabetes on nitric oxide-mediated responses in rat corpus cavernosum. European Journal of Pharmacology 376 7382. (https://doi.org/10.1016/s0014-2999(99)00347-7)

    • Search Google Scholar
    • Export Citation
  • Xie D, Odronic SI, Wu F, Pippen A, Donatucci CF & Annex BH 2007 Mouse model of erectile dysfunction due to diet-induced diabetes mellitus. Urology 70 196201. (https://doi.org/10.1016/j.urology.2007.02.060)

    • Search Google Scholar
    • Export Citation
  • Yang R, Huang YC, Lin G, Wang G, Hung S, Dai YT, Sun ZY, Lue TF & Lin CS 2009 Lack of direct androgen regulation of PDE5 expression. Biochemical and Biophysical Research Communications 380 758762. (https://doi.org/10.1016/j.bbrc.2009.01.144)

    • Search Google Scholar
    • Export Citation
  • Yeşilli C, Yaman O & Anafarta K 2001 Effect of experimental hypercholesterolemia on cavernosal structures. Urology 57 11841188. (https://doi.org/10.1016/s0090-4295(01)00974-8)

    • Search Google Scholar
    • Export Citation
  • Zvara P, Sioufi R, Schipper HM, Begin LR & Brock GB 1995 Nitric oxide mediated erectile activity is a testosterone dependent event: a rat erection model. International Journal of Impotence Research 7 209219.

    • Search Google Scholar
    • Export Citation